Tumour irradiation combined with vascular-targeted photodynamic therapy enhances antitumour effects in pre-clinical prostate cancer.

BACKGROUND: There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient 'vascular normalisation'. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP. METHODS: We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP. RESULTS: FRT induced 'vascular normalisation' changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival. CONCLUSION: Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials.

Improving deuterium metabolic imaging (DMI) signal-to-noise ratio by spectroscopic multi-echo bSSFP: A pancreatic cancer investigation.

PURPOSE: Deuterium metabolic imaging (DMI) maps the uptake of deuterated precursors and their conversion into lactate and other markers of tumor metabolism. Even after leveraging 2 H's short T1 s, DMI's signal-to-noise ratio (SNR) is limited. We hypothesize that a multi-echo balanced steady-state free precession (ME-bSSFP) approach would increase SNR compared to chemical shift imaging (CSI), while achieving spectral isolation of the metabolic precursors and products. METHODS: Suitably tuned 2 H ME-bSSFP (five echo times [TEs], ΔTE = 2.2 ms, repetition time [TR]/flip-angle = 12 ms/60°) was implemented at 15.2T and compared to CSI (TR/flip-angle = 95 ms/90°) regarding SNR and spectral isolation, in simulations, in deuterated phantoms and for the in vivo diagnosis of a mouse tumor model of pancreatic adenocarcinoma (N = 10). RESULTS: Simulations predicted an SNR increase vs. CSI of 3-5, and that the peaks of 2 H-water, 2 H6,6' -glucose, and 2 H3,3' -lactate can be well isolated by ME-bSSFP; phantoms confirmed this. In vivo, at equal spatial resolution (1.25 × 1.25 mm2 ) and scan time (10 min), 2 H6,6' -glucose's and 2 H3,3' -lactate's SNR were indeed higher for bSSFP than for CSI, three-fold for glucose (57 ± 30 vs. 19 ± 11, P < .001), doubled for water (13 ± 5 vs. 7 ± 3, P = .005). The time courses and overall localization of all metabolites agreed well, comparing CSI against ME-bSSFP. However, a clearer localization of glucose in kidneys and bladder, the detection of glucose-avid rims in certain tumors, and a heterogeneous pattern of intra-tumor lactate production could only be observed using ME-bSSFP's higher resolution. CONCLUSIONS: ME-bSSFP provides greater SNR per unit time than CSI, providing for higher spatial resolution mapping of glucose uptake and lactate production in tumors.

Deuterium MRSI characterizations of glucose metabolism in orthotopic pancreatic cancer mouse models.

Detecting and mapping metabolism in tissues represents a major step in detecting, characterizing, treating and understanding cancers. Recently introduced deuterium metabolic imaging techniques could offer a noninvasive route for the metabolic imaging of animals and humans, based on using 2 H magnetic resonance spectroscopic imaging (MRSI) to detect the uptake of deuterated glucose and the fate of its metabolic products. In this study, 2 H6,6' -glucose was administered to mice cohorts that had been orthotopically implanted with two different models of pancreatic ductal adenocarcinoma (PDAC), involving PAN-02 and KPC cell lines. As the tumors grew, 2 H6,6' -glucose was administered as bolii into the animals' tail veins, and 2 H MRSI images were recorded at 15.2 T. 2D phase-encoded chemical shift imaging experiments could detect a signal from this deuterated glucose immediately after the bolus injection for both the PDAC models, reaching a maximum in the animals' tumors ~ 20 min following administration, and nearly total decay after ~ 40 min. The main metabolic reporter of the cancers was the 2 H3,3' -lactate signal, which MRSI could detect and localize on the tumors when these were 5 mm or more in diameter. Lactate production time traces varied slightly with the animal and tumor model, but in general lactate peaked at times of 60 min or longer following injection, reaching concentrations that were ~ 10-fold lower than those of the initial glucose injection. This 2 H3,3' -lactate signal was only visible inside the tumors. 2 H-water could also be detected as deuterated glucose's metabolic product, increasing throughout the entire time course of the experiment from its ≈10 mM natural abundance background. This water resonance could be imaged throughout the entire abdomen of the animals, including an enhanced presence in the tumor, but also in other organs like the kidney and bladder. These results suggest that deuterium MRSI may serve as a robust, minimally invasive tool for the monitoring of metabolic activity in pancreatic tumors, capable of undergoing clinical translation and supporting decisions concerning treatment strategies. Comparisons with in vivo metabolic MRI experiments that have been carried out in other animal models are presented and their differences/similarities are discussed.

Identification of variable stages in murine pancreatic tumors by a multiparametric approach employing hyperpolarized 13 C MRSI, 1 H diffusivity and 1 H T1 MRI.

This study explored the usefulness of multiple quantitative MRI approaches to detect pancreatic ductal adenocarcinomas in two murine models, PAN-02 and KPC. Methods assayed included 1 H T1 and T2 measurements, quantitative diffusivity mapping, magnetization transfer (MT) 1 H MRI throughout the abdomen and hyperpolarized 13 C spectroscopic imaging. The progress of the disease was followed as a function of its development; studies were also conducted for wildtype control mice and for mice with induced mild acute pancreatitis. Customized methods developed for scanning the motion- and artifact-prone mice abdomens allowed us to obtain quality 1 H images for these targeted regions. Contrasts between tumors and surrounding tissues, however, were significantly different. Anatomical images, T2 maps and MT did not yield significant contrast unless tumors were large. By contrast, tumors showed statistically lower diffusivities than their surroundings (≈8.3 ± 0.4 x 10-4 for PAN-02 and ≈10.2 ± 0.6 x 10-4 for KPC vs 13 ± 1 x 10-3 mm2 s-1 for surroundings), longer T1 relaxation times (≈1.44 ± 0.05 for PAN-02 and ≈1.45 ± 0.05 for KPC vs 0.95 ± 0.10 seconds for surroundings) and significantly higher lactate/pyruvate ratios by hyperpolarized 13 C MR (0.53 ± 0.2 for PAN-02 and 0.78 ± 0.2 for KPC vs 0.11 ± 0.04 for control and 0.31 ± 0.04 for pancreatitis-bearing mice). Although the latter could also distinguish early-stage tumors from healthy animal controls, their response was similar to that in our pancreatitis model. Still, this ambiguity could be lifted using the 1 H-based reporters. If confirmed for other kinds of pancreatic tumors this means that these approaches, combined, can provide a route to an early detection of pancreatic cancer.

Lightweight breast implants: a novel solution for breast augmentation and reconstruction mammaplasty.

Breast augmentation and reconstruction mammaplasty have been in practice for decades and are highly prevalent surgeries performed worldwide. While overall patient satisfaction is high, common long-term effects include breast tissue atrophy, accelerated ptosis and inframammary fold breakdown. Increasing evidence attributes these events to the durative loading and compressive forces introduced by the breast implants. Mechanical challenges exceeding the elastic capacity of the breast tissue components, eventually lead to irreversible tissue stretching, directly proportional to the introduced mass. Thus, it is suggested that, contrary to long-standing dogmas, implant weight, rather than its volume, stands at the basis of future tissue compromise and deformation. A novel lightweight implant has been developed to address the drawbacks of traditional breast implants, which demonstrate equivalence between their size and weight. The B-Lite(®) breast implant (G&G Biotechnology Ltd., Haifa, Israel) design allows for a reduction in implant weight of up to 30%, while maintaining the size, form, and function of traditional breast implants. The CE-marked device can be effectively implanted using standard of care procedures and has been established safe for human use. Implantation of the B-Lite(®) breast implant is projected to significantly reduce the inherent strains imposed by standard implants, thereby conserving tissue stability and integrity over time. In summary, this novel, lightweight breast implant promises to reduce breast tissue compromise and deformation and subsequent reoperation, further improving patient safety and satisfaction.

Comparative study of immune response to local tumor destruction modalities in a murine breast cancer model.

Introduction: Immunotherapy is revolutionizing the management of multiple cancer types. However, only a subset of patients responds to immunotherapy. One mechanism of resistance is the absence of immune infiltrates within the tumor. In situ vaccine with local means of tumor destruction that can induce immunogenic cell death have been shown to enhance tumor T cell infiltration and increase efficacy of immune checkpoint blockade. Methods: Here, we compare three different forms of localize tumor destruction therapies: radiation therapy (RT), vascular targeted photodynamic therapy (VTP) and cryoablation (Cryo), which are known to induce immunogenic cell death, with their ability to induce local and systemic immune responses in a mouse 4T1 breast cancer model. The effects of combining RT, VTP, Cryo with anti-PD1 was also assessed. Results: We observed that RT, VTP and Cryo significantly delayed tumor growth and extended overall survival. In addition, they also induced regression of non-treated distant tumors in a bilateral model suggesting a systemic immune response. Flow cytometry showed that VTP and Cryo are associated with a reduction in CD11b+ myeloid cells (granulocytes, monocytes, and macrophages) in tumor and periphery. An increase in CD8+ T cell infiltration into tumors was observed only in the RT group. VTP and Cryo were associated with an increase in CD4+ and CD8+ cells in the periphery. Conclusion: These data suggest that cell death induced by VTP and Cryo elicit similar immune responses that differ from local RT.

Antitumor immunity promoted by vascular occluding therapy: lessons from vascular-targeted photodynamic therapy (VTP).

The development of cancer is tightly related to the successful evasion of neoplastic tissue from immune system surveillance, which represents a key obstacle in tumor therapy. Most conventional therapies (surgery, chemotherapy and radiation) target the tumor cells directly or indirectly, while immunotherapy attempts to enhance host anti-tumor response. In a manner similar to surgery, photodynamic therapy (PDT), also a local tumor therapy, aims at tumor ablation in its initial acute phase. Treatment success is mainly determined by tumor eradication and the absence of local recurrences. However, experience gained over several decades of therapeutic application has repeatedly hinted at long term therapeutic effects of PDT, suggesting activation of the immune system by this treatment modality. Such contribution of the immune system to treatment success was widely confirmed in many laboratories in various preclinical and some clinical studies. In this present short review, we wish to present our modest contribution to this potential therapeutic trend describing the immune response upon application of a novel photosensitizing methodology: vascular targeted photodynamic therapy (VTP) developed in our laboratories. This modality differs from classical PDT in most aspects (sensitizer: Pd-bacteriochlorophyll and consequent spectral wavelength in the near infrared, the generated photochemistry, the treatment target, treatment objective, treatment protocol and more). For example in contrast to the tumor cells that constitute the target of classical PDT, the targets of VTP are the tumor-feeding arteries and draining veins whose almost instant occlusion (minutes) leads to tumor blood stasis and eradication. Some of the mechanistic features of the induced immune response, such as innate and acquired cellular and humoral mediators, induction of new antigens, resulting from oxidative modifications and implications for anti-tumor vaccination in this different treatment environment, are discussed. VTP is about to enter phase III clinical trials for the therapy of prostate cancer and the potential involvement of the immune system may contribute an interesting aspect for the understanding and future development of this treatment modality.

Decreased Riboflavin Impregnation Time Does Not Increase the Risk for Endothelial Phototoxicity During Corneal Cross-Linking.

Purpose: To evaluate the riboflavin (RF) concentration and distribution in the corneal stroma and the risk for endothelial photodamage during corneal crosslinking (CXL) following 10- and 30-minute impregnation. Methods: De-epithelialized rabbit corneas were subjected to impregnation for 10 and 30 minutes with different RF formulations. Human corneal endothelial cells (HCECs) were subjected to different RF concentrations and ultraviolet A (UVA) dosages. Assays included fluorescence imaging, absorption spectroscopy of corneal buttons and anterior chamber humor, and cell viability staining. Results: After 10 and 30 minutes of impregnation, respectively, anterior chamber fluid showed an RF concentration of (1.6 ± 0.21)•10-4% and (5.4 ± 0.21)•10-4%, and trans-corneal absorption reported an average corneal RF concentration of 0.0266% and 0.0345%. This results in a decrease in endothelial RF concentration from 0.019% to 0.0056%, whereas endothelial UVA irradiance increases by 1.3-fold when changing from 30 to 10 minutes of impregnation. HCEC viability in cultures exposed to UVA illumination and RF concentrations as concluded for the endothelium after 10- and 30-minute impregnation was nonstatistically different at 51.0% ± 3.9 and 41.3 ± 5.0%, respectively. Conclusions: The risk for endothelial damage in CXL by RF/UVA treatment does not increase by shortened impregnation because the 30% increase in light intensity is accompanied by a 3.4-fold decrease of the RF concentration in the posterior stroma. This is substantiated by similar endothelial cell toxicity seen in vitro, which in fact appears to favor 10-minute impregnation. Translational Relevance: This study offers compelling arguments for (safely) shortening RF impregnation duration, reducing patients' burden and costly operation room time.

Systemic antitumor protection by vascular-targeted photodynamic therapy involves cellular and humoral immunity.

Vascular-targeted photodynamic therapy (VTP) takes advantage of intravascular excitation of a photosensitizer (PS) to produce cytotoxic reactive oxygen species (ROS). These ROS are potent mediators of vascular damage inducing rapid local thrombus formation, vascular occlusion, and tissue hypoxia. This light-controlled process is used for the eradication of solid tumors with Pd-bacteriochlorophyll derivatives (Bchl) as PS. Unlike classical photodynamic therapy (PDT), cancer cells are not the primary target for VTP but instead are destroyed by treatment-induced oxygen deprivation. VTP initiates acute local inflammation inside the illuminated area accompanied by massive tumor tissue death. Consequently, in the present study, we addressed the possibility of immune response induction by the treatment that may be considered as an integral part of the mechanism of VTP-mediated tumor eradication. The effect of VTP on the host immune system was investigated using WST11, which is now in phase II clinical trials for age-related macular degeneration and intended to be evaluated for cancer therapy. We found that a functional immune system is essential for successful VTP. Long-lasting systemic antitumor immunity was induced by VTP involving both cellular and humoral components. The antitumor effect was cross-protective against mismatched tumors, suggesting VTP-mediated production of overlapping tumor antigens, possibly from endothelial origin. Based on our findings we suggest that local VTP might be utilized in combination with other anticancer therapies (e.g., immunotherapy) for the enhancement of host antitumor immunity in the treatment of both local and disseminated disease.

Prompt assessment of WST11-VTP outcome using luciferase transfected tumors enables second treatment and increase in overall therapeutic rate.

This study hypothesized that success rate assessment of vascular targeted photodynamic therapy (VTP) of solid tumors 24 h post-treatment may allow prompt administration of a second treatment in case of failure, increasing the overall success rate. Here, we show that treatment of luciferase transfected CT26-luc mouse colon carcinoma tumors in BALB/c mice by VTP with WST11 (a Pd-bacteriochlorophyll-based photosensitizer) allows fast assessment of treatment success 24 h post-treatment, using bioluminescence imaging (BLI). WST11-VTP was found to abolish luciferin bioluminescence in the treated tumors resulting in two types of responses. One, comprising 75% of the mice, signified successful outcome, presenting neither BLI signal nor tumor regrowth (24 h-90 days post-VTP). The second (the remaining 25% of the mice) signified treatment failure, presenting various levels of BLI signal with subsequent tumor regrowth (24 h-90 days). Consequently, the mice that failed the first treatment were treated again. We show that treatment success rate in both VTP sessions was identical and that the cumulative success rate of the treatment increased from 75% to over 90%. These results therefore, present a fast method of assessing VTP outcome and support the feasibility of successive multiple treatments with these sensitizers in the clinical arena. The presented methodology can also be helpful in future preclinical studies, and expedite the development of VTP drugs.

Bypass of tumor drug resistance by antivascular therapy.

Multidrug resistance (MDR) presents a major obstacle for the successful chemotherapy of cancer. Its emergence during chemotherapy is attributed to a selective process, which gives a growth advantage to MDR cells within the genetically unstable neoplastic cell population. The pleiotropic nature of clinical MDR poses a great difficulty for the development of treatment strategies that aim at blocking MDR at the tumor cell level. Targeting treatment to the nonmalignant vascular network-the lifeline of the tumor-is a promising alternative for the treatment of drug-resistant tumors. The present study demonstrates that MDR in cancer can be successfully circumvented by photodynamic therapy (PDT) using an antivascular treatment protocol. We show that, although P-glycoprotein-expressing human HT29/MDR colon carcinoma cells in culture are resistant to PDT with Pd-bacteriopheophorbide (TOOKAD), the same treatment induces tumor necrosis with equal efficacy (88% vs 82%) in HT29/MDR-derived xenografts and their wild type counterparts, respectively. These results are ascribed to the rapid antivascular effects of the treatment, supporting the hypothesis that MDR tumors can be successfully eradicated by indirect approaches that bypass their inherent drug resistance. We suggest that with progress in ongoing clinical trials, TOOKAD-PDT may offer a novel option for local treatment of MDR tumors.

Ear swelling test by using laser speckle imaging with a long exposure time.

Laser speckle imaging with long exposure time has been applied noninvasively to visualize the immediate reaction of cutaneous vessels in mice in response to a known primary irritant and potential allergen­methyl salicylate. The compound has been used topically on the surface of the pinna and the reaction of the vascular network was examined. We demonstrate that irritant-induced acute vascular reaction can be effectively and accurately detected by laser speckle imaging technique. The current approach holds a great promise for application in routine screening of the cutaneous vascular response induced by contact agents, screenings of mouse ear swelling test, and testing the allergenic potential of new synthetic materials and healthcare pharmaceutical products.