Engineering Metabolism of Chimeric Antigen Receptor (CAR) Cells for Developing Efficient Immunotherapies.

Chimeric antigen receptor (CAR) T cell-based therapies have shown tremendous advancement in clinical and pre-clinical studies for the treatment of hematological malignancies, such as the refractory of pre-B cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), and large B cell lymphoma (LBCL). However, CAR T cell therapy for solid tumors has not been successful clinically. Although, some research efforts, such as combining CARs with immune checkpoint inhibitor-based therapy, have been used to expand the application of CAR T cells for the treatment of solid tumors. Importantly, further understanding of the coordination of nutrient and energy supplies needed for CAR T cell expansion and function, especially in the tumor microenvironment (TME), is greatly needed. In addition to CAR T cells, there is great interest in utilizing other types of CAR immune cells, such as CAR NK and CAR macrophages that can infiltrate solid tumors. However, the metabolic competition in the TME between cancer cells and immune cells remains a challenge. Bioengineering technologies, such as metabolic engineering, can make a substantial contribution when developing CAR cells to have an ability to overcome nutrient-paucity in the solid TME. This review introduces technologies that have been used to generate metabolically fit CAR-immune cells as a treatment for hematological malignancies and solid tumors, and briefly discusses the challenges to treat solid tumors with CAR-immune cells.

Metabolite releasing polymers control dendritic cell function by modulating their energy metabolism.

Metabolites control immune cell functions, and delivery of these metabolites in a sustained manner may be able to modulate function of the immune cells. In this study, alpha-ketoglutarate (aKG) and diol based polymeric-microparticles (termed paKG MPs) were synthesized to provide sustained release of aKG and promote an immunosuppressive cellular phenotype. Notably, after association with dendritic cells (DCs), paKG MPs modulated the intracellular metabolic-profile/pathways, and decreased glycolysis and mitochondrial respiration in vitro. These metabolic changes resulted in modulation of MHC-II, CD86 expression in DCs, and altered the frequency of regulatory T cells (Tregs), and T-helper type-1/2/17 cells in vitro. This unique strategy of intracellular delivery of key-metabolites in a sustained manner provides a new direction in immunometabolism field-based immunotherapy with potential applications in different diseases associated with immune disorders.

Mouse Model of Thyroid Cancer Progression and Dedifferentiation Driven by STRN-ALK Expression and Loss of p53: Evidence for the Existence of Two Types of Poorly Differentiated Carcinoma.

Background: Thyroid tumor progression from well-differentiated cancer to poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) involves step-wise dedifferentiation associated with loss of iodine avidity and poor outcomes. ALK fusions, typically STRN-ALK, are found with higher incidence in human PDTC compared with well-differentiated cancer and, as previously shown, can drive the development of murine PDTC. The aim of this study was to evaluate thyroid cancer initiation and progression in mice with concomitant expression of STRN-ALK and inactivation of the tumor suppressor p53 (Trp53) in thyroid follicular cells. Methods: Transgenic mice with thyroid-specific expression of STRN-ALK and biallelic p53 loss were generated and aged on a regular diet or with methimazole and sodium perchlorate goitrogen treatment. Development and progression of thyroid tumors were monitored by using ultrasound imaging, followed by detailed histological and immunohistochemical evaluation. Gene expression analysis was performed on selected tumor samples by using RNA-Seq and quantitative RT-PCR. Results: In mice treated with goitrogen, the first thyroid cancers appeared at 6 months of age, reaching 86% penetrance by the age of 12 months, while a similar rate (71%) of tumor occurrence in mice on regular diet was observed by 18 months of age. Histological examination revealed well-differentiated papillary thyroid carcinomas (PTC) (n = 26), PDTC (n = 21), and ATC (n = 8) that frequently coexisted in the same thyroid gland. The tumors were frequently lethal and associated with the development of lung metastasis in 24% of cases. Histological and immunohistochemical characteristics of these cancers recapitulated tumors seen in humans. Detailed analysis of PDTC revealed two tumor types with distinct cell morphology and immunohistochemical characteristics, designated as PDTC type 1 (PDTC1) and type 2 (PDTC2). Gene expression analysis showed that PDTC1 tumors retained higher expression of thyroid differentiation genes including Tg and Slc5a5 (Nis) as compared with PDTC2 tumors. Conclusions: In this study, we generated a new mouse model of multistep thyroid cancer dedifferentiation with evidence of progression from PTC to PDTC and ATC. Further, PDTC in these mice showed two distinct histologic appearances correlated with levels of expression of thyroid differentiation and iodine metabolism genes, suggesting a possibility of existence of two PDTC types with different functional characteristics and potential implication for therapeutic approaches to these tumors.

Exogenous sickle erythrocytes combined with vascular disruption trigger disseminated tumor vaso-occlusion and lung tumor regression.

Hypoxic tumor niches are chief causes of treatment resistance and tumor recurrence. Sickle erythrocytes' (SSRBCs') intrinsic oxygen-sensing functionality empowers them to access such hypoxic niches wherein they form microaggregates that induce focal vessel closure. In search of measures to augment the scale of SSRBC-mediated tumor vaso-occlusion, we turned to the vascular disrupting agent, combretastatin A-4 (CA-4). CA-4 induces selective tumor endothelial injury, blood stasis, and hypoxia but fails to eliminate peripheral tumor foci. In this article, we show that introducing deoxygenated SSRBCs into tumor microvessels treated with CA-4 and sublethal radiation (SR) produces a massive surge of tumor vaso-occlusion and broadly propagated tumor infarctions that engulfs treatment-resistant hypoxic niches and eradicates established lung tumors. Tumor regression was histologically corroborated by significant treatment effect. Treated tumors displayed disseminated microvessels occluded by tightly packed SSRBCs along with widely distributed pimidazole-positive hypoxic tumor cells. Humanized HbS-knockin mice (SSKI) but not HbA-knockin mice (AAKI) showed a similar treatment response underscoring SSRBCs as the paramount tumoricidal effectors. Thus, CA-4-SR-remodeled tumor vessels license SSRBCs to produce an unprecedented surge of tumor vaso-occlusion and infarction that envelops treatment-resistant tumor niches resulting in complete tumor regression. Strategically deployed, these innovative tools constitute a major conceptual advance with compelling translational potential.

Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis.

cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling. Here, we introduce a new fusion protein consisting of a light-activated adenylyl cyclase (bPAC) and luciferase (nLuc). This construct allows dual activation of cAMP production through temporally precise photostimulation or chronic chemical stimulation that can be fine-tuned to mimic physiological levels and duration of cAMP synthesis to trigger downstream events. By targeting this construct to different compartments, we show that cAMP produced in the cytosol and nucleus stimulates proliferation in thyroid cells. The bPAC-nLuc fusion construct adds a new reagent to the available toolkit to study cAMP-regulated processes in living cells.

Mouse Model of Poorly Differentiated Thyroid Carcinoma Driven by STRN-ALK Fusion.

Chromosomal rearrangements of the ALK gene, which lead to constitutive activation of ALK tyrosine kinase, are found in various cancers. In thyroid cancers, ALK fusions, most commonly the STRN-ALK fusion, are detected in papillary thyroid cancer and with higher frequency in poorly differentiated and anaplastic thyroid cancers. Our aim was to establish a mouse model of thyroid-specific expression of STRN-ALK and to test whether this fusion drives the development of thyroid cancer with a propensity for dedifferentiation. Transgenic Tg-STRN-ALK mice with thyroglobulin-controlled expression of STRN-ALK were generated and aged with or without goitrogen treatment. Thyroids from these mice were subjected to histologic and immunohistochemical analysis. Transgenic mice with thyroid-specific expression of STRN-ALK developed poorly differentiated thyroid tumors by the age of 12 months in 22% of mice without goitrogen treatment and in 36% of mice with goitrogen treatment. Histologically and immunohistochemically, the tumors resembled poorly differentiated thyroid cancers in humans, demonstrating a solid growth pattern with sheets of round or spindle-shaped cells, decreased expression of thyroglobulin, and a tendency to lose E-cadherin. In this study, we report a novel mouse model of poorly differentiated thyroid cancer driven by the STRN-ALK oncogene with phenotypic features closely recapitulating human tumor, and with a more pronounced phenotype after additional thyroid-stimulating hormone stimulation.

Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase.

Rap1 proteins are members of the Ras subfamily of small GTPases involved in many biological responses, including adhesion, cell proliferation, and differentiation. Like all small GTPases, they work as molecular allosteric units that are active in signaling only when associated with the proper membrane compartment. Prenylation, occurring in the cytosol, is an enzymatic posttranslational event that anchors small GTPases at the membrane, and prenyl-binding proteins are needed to mask the cytoplasm-exposed lipid during transit to the target membrane. However, several of these proteins still await discovery. In this study, we report that cyclase-associated protein 1 (CAP1) binds Rap1. We found that this binding is GTP-independent, does not involve Rap1's effector domain, and is fully contained in its C-terminal hypervariable region (HVR). Furthermore, Rap1 prenylation was required for high-affinity interactions with CAP1 in a geranylgeranyl-specific manner. The prenyl binding specifically involved CAP1's C-terminal hydrophobic ß-sheet domain. We present a combination of experimental and computational approaches, yielding a model whereby the high-affinity binding between Rap1 and CAP1 involves electrostatic and nonpolar side-chain interactions between Rap1's HVR residues, lipid, and CAP1 ß-sheet domain. The binding was stabilized by the lipid insertion into the ß-solenoid whose interior was occupied by nonpolar side chains. This model was reminiscent of the recently solved structure of the PDEδ-K-Ras complex; accordingly, disruptors of this complex, e.g. deltarasin, blocked the Rap1-CAP1 interaction. These findings indicate that CAP1 is a geranylgeranyl-binding partner of Rap1.

De novo cerebrovascular malformation in the adult mouse after endothelial Alk1 deletion and angiogenic stimulation.

BACKGROUND AND PURPOSE: In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor stimulation induces brain AVMs in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal vascular endothelial growth factor stimulation is sufficient to induce brain AVM in the adult mouse. METHODS: Focal angiogenesis was induced in the brain of 8-week-old Pdgfb-iCreER;Alk1(2f/2f) mice by injection of adeno-associated viral vectors expressing vascular endothelial growth factor. Two weeks later, EC-Alk1 deletion was induced by tamoxifen treatment. Vascular morphology was analyzed, and EC proliferation and dysplasia index (number of vessels with diameter>15 µm per 200 vessels) were quantified 10 days after tamoxifen administration. RESULTS: Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of tamoxifen-treated Pdgfb-iCreER;Alk1(2f/2f) mice. Induced brain AVMs were marked by increased dysplasia index (P<0.001) and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs. CONCLUSIONS: Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo brain AVM.

Magnetic nanoparticles: an emerging technology for malignant brain tumor imaging and therapy.

Magnetic nanoparticles (MNPs) represent a promising nanomaterial for the targeted therapy and imaging of malignant brain tumors. Conjugation of peptides or antibodies to the surface of MNPs allows direct targeting of the tumor cell surface and potential disruption of active signaling pathways present in tumor cells. Delivery of nanoparticles to malignant brain tumors represents a formidable challenge due to the presence of the blood-brain barrier and infiltrating cancer cells in the normal brain. Newer strategies permit better delivery of MNPs systemically and by direct convection-enhanced delivery to the brain. Completion of a human clinical trial involving direct injection of MNPs into recurrent malignant brain tumors for thermotherapy has established their feasibility, safety and efficacy in patients. Future translational studies are in progress to understand the promising impact of MNPs in the treatment of malignant brain tumors.

Spectral imaging reveals microvessel physiology and function from anastomoses to thromboses.

Abnormal microvascular physiology and function is common in many diseases. Numerous pathologies include hypervascularity, aberrant angiogenesis, or abnormal vascular remodeling among the characteristic features of the disease, and quantitative imaging and measurement of microvessel function can be important to increase understanding of these diseases. Several optical techniques are useful for direct imaging of microvascular function. Spectral imaging is one such technique that can be used to assess microvascular oxygen transport function with high spatial and temporal resolution in microvessel networks through measurements of hemoglobin saturation. We highlight novel observation made with our intravital microscopy spectral imaging system employed with mouse dorsal skin-fold window chambers for imaging hemoglobin saturation in microvessel networks. Specifically, we image acute oxygenation fluctuations in a tumor microvessel network, the development of arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia, and the formation of spontaneous and induced microvascular thromboses and occlusions.

In vivo functional differences in microvascular response of 4T1 and Caki-1 tumors after treatment with OXi4503.

4T1 mouse mammary adenocarcinomas and Caki-1 human renal cell carcinomas grown in mouse dorsal window chambers were serially treated with the vascular disrupting agent (VDA) OXi4503 and their responses compared. The real-time in vivo response was assessed using spectral imaging of microvascular hemoglobin saturation. To our knowledge this is the first use of spectral imaging technology for investigation of vascular disrupting agents. Previous research showing tumor size dependence in the treatment response to VDAs suggested that for the size of tumors used in this study only a moderate response would be observed; however, the tumors unexpectedly had very different responses to treatment. Caki-1 tumors showed little permanent vessel damage and experienced transient vessel collapse with time-dependent oxygenation changes followed by recovery starting at 6 h after treatment. Caki-1 tumors did not manifest necrotic avascular regions even after repeated treatments. These results are consistent with those obtained using other imaging modalities and histology. In contrast, similarly sized 4T1 tumors showed extensive vessel disintegration, minor vascular collapse, and a drop in tumor oxygenation up to 6 h post-treatment, after which reperfusion of collapsed vessels and extensive vascular remodeling and neovascularization of the tumor rim occurred from 8-48 h. The completely disintegrated vessels did not recover and left behind avascular and apparently necrotic regions in the tumor core. Spectral imaging appears to be a useful technique for in vivo investigation of vascular disrupting agents. The differential responses of these two tumor-types suggest that further investigation of the mechanisms of action of VDAs and individual characterization of tumor VDA-responses may be needed for optimal clinical use of these agents.

ACE2 activation promotes antithrombotic activity.

The aim of the present study was to test the hypothesis that the activation of the angiotensin-converting enzyme (ACE)2/angiotensin-(1-7)/Mas receptor axis by use of a novel ACE2 activator (XNT) would protect against thrombosis. Thrombi were induced in the vena cava of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats, and ACE2 and ACE activity in the thrombus was determined. Real-time thrombus formation was viewed through intravital microscopy of vessels in nude mice. Thrombus weight was 40% greater in the SHR (4.99 +/- 0.39 versus 7.04 +/- 0.66 mg). This weight increase was associated with a 20% decrease in ACE2 activity in the thrombus. In contrast, there were no differences between the WKY and SHR in ACE2 protein and ACE activity in the thrombi. ACE2 inhibition (DX600; 0.1 micromol/L/kg) increased thrombus weight by 30% and XNT treatment (10 mg/kg) resulted in a 30% attenuation of thrombus formation in the SHR. Moreover, XNT reduced platelet attachment to injured vessels, reduced thrombus size, and prolonged the time for complete vessel occlusion in mice. Thus, a decrease in thrombus ACE2 activity is associated with increased thrombus formation in SHR. Furthermore, ACE2 activation attenuates thrombus formation and reduces platelet attachment to vessels. These results suggest that ACE2 could be a novel target for the treatment of thrombogenic diseases.

Real-time imaging of de novo arteriovenous malformation in a mouse model of hereditary hemorrhagic telangiectasia.

Arteriovenous malformations (AVMs) are vascular anomalies where arteries and veins are directly connected through a complex, tangled web of abnormal arteries and veins instead of a normal capillary network. AVMs in the brain, lung, and visceral organs, including the liver and gastrointestinal tract, result in considerable morbidity and mortality. AVMs are the underlying cause of three major clinical symptoms of a genetic vascular dysplasia termed hereditary hemorrhagic telangiectasia (HHT), which is characterized by recurrent nosebleeds, mucocutaneous telangiectases, and visceral AVMs and caused by mutations in one of several genes, including activin receptor-like kinase 1 (ALK1). It remains unknown why and how selective blood vessels form AVMs, and there have been technical limitations to observing the initial stages of AVM formation. Here we present in vivo evidence that physiological or environmental factors such as wounds in addition to the genetic ablation are required for Alk1-deficient vessels to develop to AVMs in adult mice. Using the dorsal skinfold window chamber system, we have demonstrated for what we believe to be the first time the entire course of AVM formation in subdermal blood vessels by using intravital bright-field images, hyperspectral imaging, fluorescence recordings of direct arterial flow through the AV shunts, and vascular casting techniques. We believe our data provide novel insights into the pathogenetic mechanisms of HHT and potential therapeutic approaches.

Multimodal optical imaging of microvessel network convective oxygen transport dynamics.

Convective oxygen transport by microvessels depends on several parameters, including red blood cell flux and oxygen saturation. We demonstrate the use of intravital microscopy techniques to measure hemoglobin saturations, red blood cell fluxes and velocities, and microvessel cross-sectional areas in regions of microvascular networks containing multiple vessels. With these methods, data can be obtained at high spatial and temporal resolution and correlations between oxygen transport and hemodynamic parameters can be assessed. In vivo data are presented for a mouse mammary adenocarcinoma grown in a dorsal skinfold window chamber model.