Mukund Seshadri

Non-invasive multimodal functional imaging of the intestine with frozen micellar naphthalocyanines

Overview There is a need for safer and improved methods for non-invasive imaging of the gastrointestinal tract. Modalities based on X-ray radiation, magnetic resonance and ultrasound suffer from limitations with respect to safety, accessibility or lack of adequate contrast. Functional intestinal imaging of dynamic gut processes has not been practical using existing approaches. Here, we report the development of a family of nanoparticles that can withstand the harsh conditions of the stomach and intestine, avoid systemic absorption, and give rise to good optical contrast for photoacoustic imaging. The hydrophobicity of naphthalocyanine dyes was exploited to generate purified ~20 nm frozen micelles, which we call nanonaps, with tunable and large near-infrared absorption values (>1000). Unlike conventional chromophores, nanonaps exhibited non-shifting spectra at ultrahigh optical densities and, following oral administration in mice, passed safely through the gastrointestinal tract. Non-invasive, non-ionizing photoacoustic techniques were used to visualize nanonap intestinal distribution with low background and remarkable resolution with 0.5 cm depth, and enabled real-time intestinal functional imaging with ultrasound co-registration. Positron emission tomography following seamless nanonap radiolabelling allowed complementary whole body imaging.

Rb1 and Trp53 cooperate to suppress prostate cancer lineage plasticity, metastasis, and antiandrogen resistance

Evading cancer drugs by identity fraud Prostate cancer growth is fueled by male hormones called androgens. Drugs targeting the androgen receptor (AR) are initially efficacious, but most tumors eventually become resistant (see the Perspective by Kelly and Balk). Mu et al. found that prostate cancer cells escaped the effects of androgen deprivation therapy through a change in lineage identity. Functional loss of the tumor suppressors TP53 and RB1 promoted a shift from AR-dependent luminal epithelial cells to AR-independent basal-like cells. In related work, Ku et al. found that prostate cancer metastasis, lineage switching, and drug resistance were driven by the combined loss of the same tumor suppressors and were accompanied by increased expression of the epigenetic regulator Ezh2. Ezh2 inhibitors reversed the lineage switch and restored sensitivity to androgen deprivation therapy in experimental models. Science, this issue p. 84, p. 78; see also p. 29 Prostate cancer cells escape androgen deprivation therapy by morphing into a cell type that does not require androgens. Prostate cancer relapsing from antiandrogen therapies can exhibit variant histology with altered lineage marker expression, suggesting that lineage plasticity facilitates therapeutic resistance. The mechanisms underlying prostate cancer lineage plasticity are incompletely understood. Studying mouse models, we demonstrate that Rb1 loss facilitates lineage plasticity and metastasis of prostate adenocarcinoma initiated by Pten mutation. Additional loss of Trp53 causes resistance to antiandrogen therapy. Gene expression profiling indicates that mouse tumors resemble human prostate cancer neuroendocrine variants; both mouse and human tumors exhibit increased expression of epigenetic reprogramming factors such as Ezh2 and Sox2. Clinically relevant Ezh2 inhibitors restore androgen receptor expression and sensitivity to antiandrogen therapy. These findings uncover genetic mutations that enable prostate cancer progression; identify mouse models for studying prostate cancer lineage plasticity; and suggest an epigenetic approach for extending clinical responses to antiandrogen therapy.

Role of Vitamin D Receptor in the Antiproliferative Effects of Calcitriol in Tumor-Derived Endothelial Cells and Tumor Angiogenesis In vivo

Calcitriol (1,25-dihydroxycholecalciferol), the major active form of vitamin D, is antiproliferative in tumor cells and tumor-derived endothelial cells (TDEC). These actions of calcitriol are mediated at least in part by vitamin D receptor (VDR), which is expressed in many tissues including endothelial cells. To investigate the role of VDR in calcitriol effects on tumor vasculature, we established TRAMP-2 tumors subcutaneously into either VDR wild-type (WT) or knockout (KO) mice. Within 30 days post-inoculation, tumors in KO mice were larger than those in WT (P < 0.001). TDEC from WT expressed VDR and were able to transactivate a reporter gene whereas TDEC from KO mice were not. Treatment with calcitriol resulted in growth inhibition in TDEC expressing VDR. However, TDEC from KO mice were relatively resistant, suggesting that calcitriol-mediated growth inhibition on TDEC is VDR-dependent. Further analysis of the TRAMP-C2 tumor sections revealed that the vessels in KO mice were enlarged and had less pericyte coverage compared with WT (P < 0.001). Contrast-enhanced magnetic resonance imaging showed an increase in vascular volume of TRAMP tumors grown in VDR KO mice compared with WT mice (P < 0.001) and FITC-dextran permeability assay suggested a higher extent of vascular leakage in tumors from KO mice. Using ELISA and Western blot analysis, there was an increase of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, angiopoietin 1, and platelet-derived growth factor-BB levels observed in tumors from KO mice. These results indicate that calcitriol-mediated antiproliferative effects on TDEC are VDR-dependent and loss of VDR can lead to abnormal tumor angiogenesis.

Targeting CXCL12/CXCR4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases

Significance Novel advances in viral oncotherapy require effective direct oncolysis and manipulation of the tumor microenvironment, which has proven to be an important target in cancer treatment. The CXCR4 receptor for the CXCL12 chemokine is one of the key stimuli involved in signaling interactions between tumor cells and their microenvironment, suggesting that inhibition of this pathway by oncolytic viruses expressing the CXCR4 antagonist should increase efficacy over that mediated by oncolysis alone. We are unique in demonstrating that targeting CXCR4 signaling through an oncolytic vaccinia virus yields a significant therapeutic impact against primary and metastatic breast cancer. Oncolytic viruses hold promise for the treatment of cancer, but their interaction with the tumor microenvironment needs to be elucidated for optimal tumor cell killing. Because the CXCR4 receptor for the stromal cell-derived factor-1 (SDF-1/CXCL12) chemokine is one of the key stimuli involved in signaling interactions between tumor cells and their stromal microenvironment, we used oncolytic virotherapy with a CXCR4 antagonist to target the CXCL12/CXCR4 signaling axis in a triple-negative 4T1 breast carcinoma in syngeneic mice. We show here that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with orthotopic tumors attains higher intratumoral concentration than its soluble counterpart and exhibits increased efficacy over that mediated by oncolysis alone. A systemic delivery of the armed virus after resection of the primary tumor was efficacious in inhibiting the development of spontaneous metastasis and increased overall tumor-free survival. Inhibition of tumor growth with the armed virus was associated with destruction of tumor vasculature, reductions in expression of CXCL12 and VEGF, and decrease in intratumoral numbers of bone marrow-derived endothelial and myeloid cells. These changes led to induction of antitumor antibody responses and resistance to tumor rechallenge. Engineering an oncolytic virus armed with a CXCR4 antagonist represents an innovative strategy that targets multiple elements within the tumor microenvironment. As such, this approach could have a significant therapeutic impact against primary and metastatic breast cancer.

Tumor vascular maturation and improved drug delivery induced by methylselenocysteine leads to therapeutic synergy with anticancer drugs.

Purpose: Our previously reported therapeutic synergy between naturally occurring seleno-amino acid methylselenocysteine (MSC) and anticancer drugs could not be shown in vitro. Studies were carried out to investigate the potential role of MSC-induced tumor vascular maturation and increased drug delivery in the observed therapeutic synergy in vivo. Experimental Design: Mice bearing s.c. FaDu human head and neck squamous cell carcinoma xenografts were treated with MSC (0.2 mg/d × 14 days orally). Changes in microvessel density (CD31), vascular maturation (CD31/α-smooth muscle actin), perfusion (Hoechst 33342/DiOC7), and permeability (dynamic contrast-enhanced magnetic resonance imaging) were determined at the end of the 14-day treatment period. Additionally, the effect of MSC on drug delivery was investigated by determining intratumoral concentration of doxorubicin using high-performance liquid chromatography and fluorescence microscopy. Results: Double immunostaining of tumor sections revealed a marked reduction (∼40%) in microvessel density accompanying tumor growth inhibition following MSC treatment along with a concomitant increase in the vascular maturation index (∼30% > control) indicative of increased pericyte coverage of microvessels. Hoechst 33342/DiOC7 staining showed improved vessel functionality, and dynamic contrast-enhanced magnetic resonance imaging using the intravascular contrast agent, albumin-GdDTPA, revealed a significant reduction in vascular permeability following MSC treatment. Consistent with these observations, a 4-fold increase in intratumoral doxorubicin levels was observed with MSC pretreatment compared with administration of doxorubicin alone. Conclusion: These results show, for the first time, the antiangiogenic effects of MSC results in tumor growth inhibition, vascular maturation in vivo, and enhanced anticancer drug delivery that are associated with the observed therapeutic synergy in vivo.

Current Understanding of the Molecular Biology of Pancreatic Neuroendocrine Tumors

Pancreatic neuroendocrine tumors (PanNETs) are complicated and often deadly neoplasms. A recent increased understanding of their molecular biology has contributed to expanded treatment options. DNA sequencing of samples derived from patients with PanNETs and rare genetic syndromes such as multiple endocrine neoplasia type 1 (MEN1) and Von Hippel-Lindau (VHL) syndrome reveals the involvement of MEN1, DAXX/ATRX, and the mammalian target of rapamycin (mTOR) pathways in PanNET tumorigenesis. Gene knock-out/knock-in studies indicate that inactivation of factors including MEN1 and abnormal PI3K/mTOR signaling uncouples endocrine cell cycle progression from the control of environmental cues such as glucose, leading to islet cell overgrowth. In addition, accumulating evidence suggests that further impairment of endothelial-endocrine cell interactions contributes to tumor invasion and metastasis. Recent phase III clinical trials have shown that therapeutic interventions, such as sunitinib and everolimus, targeting those signal transduction pathways improve disease-free survival rates. Yet, cure in the setting of advanced disease remains elusive. Further advances in our understanding of the molecular mechanisms of PanNETs and improved preclinical models will assist in developing personalized therapy utilizing novel drugs to provide prolonged control or even cure the disease.

Tumor Vascular Response to Photodynamic Therapy and the Antivascular Agent 5,6-Dimethylxanthenone-4-Acetic Acid: Implications for Combination Therapy

Purpose: Photodynamic therapy (PDT) is a clinically approved treatment for a variety of solid malignancies. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a potent vascular targeting agent that has been shown to be effective against a variety of experimental rodent tumors and xenografts and is currently undergoing clinical evaluation. We have previously reported that the activity of PDT against transplanted mouse tumors is selectively enhanced by DMXAA. In the present study, we investigated the in vivo tumor vascular responses to the two treatments given alone and in combination. Experimental Design: Vascular responses to (i) four different PDT regimens using the photosensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) at two different fluences (128 and 48 J/cm2) and fluence rates (112 and 14 mW/cm2), (ii) 5-aminolevulinic acid (ALA)–sensitized PDT (135 J/cm2 at 75 mW/cm2), (iii) DMXAA at a high (30 mg/kg) and low dose (25 mg/kg), and (iv) the combination of HPPH-PDT (48 J/cm2 at 112 mW/cm2) and low-dose DMXAA were studied in BALB/c mice bearing Colon-26 tumors. Results: PDT-induced changes in vascular permeability, determined using noninvasive magnetic resonance imaging with a macromolecular contrast agent, were regimen dependent and did not predict tumor curability. However, a pattern of increasing (4 hours after treatment) and then decreasing (24 hours after) contrast agent concentrations in tumors, seen after high-dose DMXAA or the combination of PDT and low-dose DMXAA, was associated with long-term cure rates of >70%. This pattern was attributed to an initial increase in vessel permeability followed by substantial endothelial cell damage (CD31 immunohistochemistry) and loss of blood flow (fluorescein exclusion assay). Low dose–rate PDT, regardless of the delivered dose, increased the level of magnetic resonance contrast agent in peritumoral tissue, whereas treatment with either DMXAA alone, or PDT and DMXAA in combination resulted in a more selective tumor vascular response. Conclusions: The observed temporal and spatial differences in the response of tumor vessels to PDT and DMXAA treatments could provide valuable assistance in the optimization of scheduling when combining these therapies. The combination of PDT and DMXAA provides therapeutically synergistic and selective antitumor activity. Clinical evaluation of this combination is warranted.

Light Delivery Over Extended Time Periods Enhances the Effectiveness of Photodynamic Therapy

Purpose: The rate of energy delivery is a principal factor determining the biological consequences of photodynamic therapy (PDT). In contrast to conventional high-irradiance treatments, recent preclinical and clinical studies have focused on low-irradiance schemes. The objective of this study was to investigate the relationship between irradiance, photosensitizer dose, and PDT dose with regard to treatment outcome and tumor oxygenation in a rat tumor model. Experimental Design: Using the photosensitizer HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide), a wide range of PDT doses that included clinically relevant photosensitizer concentrations was evaluated. Magnetic resonance imaging and oxygen tension measurements were done along with the Evans blue exclusion assay to assess vascular response, oxygenation status, and tumor necrosis. Results: In contrast to high-incident laser power (150 mW), low-power regimens (7 mW) yielded effective tumor destruction. This was largely independent of PDT dose (drug-light product), with up to 30-fold differences in photosensitizer dose and 15-fold differences in drug-light product. For all drug-light products, the duration of light treatment positively influenced tumor response. Regimens using treatment times of 120 to 240 min showed marked reduction in signal intensity in T2-weighted magnetic resonance images at both low (0.1 mg/kg) and high (3 mg/kg) drug doses compared with short-duration (6-11 min) regimens. Significantly greater reductions in pO2 were observed with extended exposures, which persisted after completion of treatment. Conclusions: These results confirm the benefit of prolonged light exposure, identify vascular response as a major contributor, and suggest that duration of light treatment (time) may be an important new treatment variable.

CXCL12/CXCR4 Blockade by Oncolytic Virotherapy Inhibits Ovarian Cancer Growth by Decreasing Immunosuppression and Targeting Cancer-Initiating Cells

Signals mediated by the chemokine CXCL12 and its receptor CXCR4 are involved in the progression of ovarian cancer through enhancement of tumor angiogenesis and immunosuppressive networks that regulate dissemination of peritoneal metastasis and development of cancer-initiating cells (CICs). In this study, we investigated the antitumor efficacy of a CXCR4 antagonist expressed by oncolytic vaccinia virus (OVV) against an invasive variant of the murine epithelial ovarian cancer cell line ID8-T. This variant harbors a high frequency of CICs that form multilayered spheroid cells and express the hyaluronan receptor CD44, as well as stem cell factor receptor CD117 (c-kit). Using an orthotopic ID8-T tumor model, we observed that i.p. delivery of a CXCR4 antagonist–expressing OVV led to reduced metastatic spread of tumors and improved overall survival compared with oncolysis alone. Inhibition of tumor growth with the armed virus was associated with efficient killing of CICs, reduced expression of ascitic CXCL12 and vascular endothelial growth factor, and decreases in i.p. numbers of endothelial and myeloid cells, as well as plasmacytoid dendritic cells. These changes, together with reduced recruitment of T regulatory cells, were associated with higher ratios of IFN-γ+/IL-10+ tumor-infiltrating T lymphocytes, as well as induction of spontaneous humoral and cellular antitumor responses. Similarly, the CXCR4 antagonist released from virally infected human CAOV2 ovarian carcinoma cells inhibited peritoneal dissemination of tumors in SCID mice, leading to improved tumor-free survival in a xenograft model. Our findings demonstrate that OVV armed with a CXCR4 antagonist represents a potent therapy for ovarian CICs with a broad antitumor repertoire.

Photoacoustic Imaging of Vascular Hemodynamics: Validation with Blood Oxygenation Level–Dependent MR Imaging

PURPOSE To noninvasively assess vascular hemodynamics with photoacoustic imaging (PAI) and blood oxygenation level-dependent (BOLD) magnetic resonance (MR) imaging in phantoms and in an animal model. MATERIALS AND METHODS In vivo studies were performed with institutional animal care and use committee approval. In vitro experiments were performed by using a tissue-mimicking phantom in multiple oxygenation conditions (n = 6) to compare PAI measurements and BOLD MR imaging measurements. PAI and T2-weighted spin-echo-based BOLD MR imaging were performed to assess tumor response to carbogen (95% O2, 5% CO2) in mice with head and neck tumors before (n = 11) and after (n = 9) treatment with a vascular disrupting agent (VDA). Two-tailed Pearson correlation analysis was performed to determine the correlation between the parameters measured with PAI and BOLD MR imaging in vitro. Two-tailed paired t tests were used to compare change in tumor hemoglobin oxygen saturation (sO2) levels and BOLD signal in response to carbogen. Changes in PAI and BOLD signal intensity before and after VDA treatment were analyzed for significance by using analysis of variance with repeated measures. RESULTS Phantom measurements yielded good correlation between photoacoustically derived sO2 levels and BOLD signal intensity (r = 0.937, P = .005) and partial pressure of oxygen (r = 0.981, P = .005). In vivo hemodynamic response to carbogen was characterized by a significant increase in tumor sO2 levels (P = .003) and BOLD signal (P = .001). When compared with pretreatment estimates, treatment with VDA resulted in a significant reduction in the tumor hemodynamic response to carbogen at PAI (P = .030). CONCLUSION Carbogen-based functional imaging with PAI and BOLD MR imaging enables monitoring of early changes in tumor hemodynamics after vascular targeted therapy.