Dina Preise

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.

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.

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.

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.

In vivo dynamic light scattering microscopy of tumour blood vessels.

We present a study investigating the use of dynamic light scattering microscopy based on the temporal laser speckles contrast that is produced over time by red blood cells (RBCs) flowing inside tumour blood vessels. The proposed noninvasive methodology is capable of producing high‐resolution images of tumour vasculature. The technique is effective at producing images from tissue at a significant depth, as well as potentially having the ability to monitor tumour perfusion. An advantage of this methodology is that it has improved depth penetration compared with conventional imaging techniques (such as reflected‐light microscopy), and one can avoid the use of any fluorescent or artificial chemicals for labeling. This is advantageous since labeling materials can affect imaging and animal welfare with respect to experiments that require continuous and repetitive monitoring.

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.

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

Abstract. 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 allergen—methyl 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.

Palliative treatment of obstructive esophageal cancer by vascular targeted photodynamic therapy (VTP): Preclinical study in orthotopic rat model.

59 Background: Vascular targeted photodynamic therapy (VTP) based on local sensitization of circulating WST11 (Pd-bacteriochlorophyll), enables effective focal ablation of solid tumors. WST11 clears from the circulation with t1/2 ~40 min (in humans) and does not accumulate in tissues which assures high level of safety and minimal adverse effects. Here we evaluate WST11-VTP for the treatment of obstructive esophageal cancer aiming at developing clinically relevant protocol. Methods: Using endoscopy guided procedure rat esophageal cancer cells (JA) were implanted in the mid esophagus. Following intravenous infusion of WST11, established tumors were illuminated by cylindrical fiber using the same endoscope sleeve. Safety and efficacy were evaluated by monitoring animal weight shift and histology of treated esophageal tissues. Treated animals included control (light/no WST11 or WST11/no light), WST11-VTP and WST11-VTP following treatment with bevacizumab at day -3. Results: WST11-VTP on esophageal tissues res...

Abstract B012: Immunomodulated VTP enables cure of metastatic prostate and breast cancers in animal models

Vascular-targeted photodynamic therapy with WST11 (VTP) enables safe and efficient non-thermal ablation of solid tumors as demonstrated by recent phase II and III clinical trials with localized prostate cancer. Studies in animal models in our labs revealed that VTP of immunogenic tumors elicits innate/adaptive immune responses and over 80% cures with consequent long-lasting anti-tumor protection. Here we show that local VTP combined with immune modulation in rodents, bearing weakly immunogenic prostate and triple negative breast tumors, results in eradication of remote metastases and animal cure. Methodology: 4T1-luc cells were subcutaneously grafted at the hind leg of Balb/C mice. Colony forming assay confirmed tumor cell spreading to the lungs already by day 7 post grafting when local VTP was delivered to the primary tumor. The study comprised: (i) animals treated by VTP alone or (ii) following 50mg/kg cyclophosphamide (CTX) on day 4 post grafting, or (iii) VTP combined with anti-CTLA4 antibodies administered on days 1, 4, 7 and 10 post VTP according to standard protocol. Appropriate control groups using immunomodulatory agents alone or untreated tumors were utilized. Cure was defined as absence of detectable primary or metastatic tumor by necropsy at 90 days. Mat-Lu-luc prostate cancer cells were grafted orthotopically in Copenhagen rats. Surgical removal of tumor bearing prostate and follow up of disease progression demonstrated that on day 10 post grafting, the day of VTP, metastatic spread has already been initiated in 57% of animals, eventually developing abdominal and visceral metastases. Treatment groups comprised (i) animals treated by VTP with or (ii) without CTX pre-treatment followed by prostate removal three days after VTP or (iii) surgery only. Results: Treatment of primary 4T1-luc tumors with VTP resulted in a complete primary tumor ablation in 50% of treated animals. Less then 10% of animals treated with VTP alone were cured with the rest developing lung metastases, while combination with CTX resulted in 30% cures. CTX induced reduction of total cell counts in tumor draining lymph nodes (LN) and spleens on VTP day followed by cell expansion over the following four days. Significant CTX-mediated reduction of Foxp3+ cells accompanied by increase in CD8+ cells during this period could be responsible for observed anti-metastatic effect of the combined treatment. Moreover, no significant change in total CD4+ cells observed at the same time suggests expansion of T helper cell subsets with concomitant suppression of regulatory population. Anti-CTLA-4 treatment, although significantly improved primary response rates after sub-optimal VTP (56% vs 14% in combined group vs VTP), did not prevent metastases suggesting additional escape mechanisms. VTP of orthotopic prostate tumors followed by whole gland removal resulted in 45% metastatic rate vs 57% in group treated by surgery alone on the same day. Pre-treatment with 50mg/kg CTX three days prior to VTP reduced metastatic rate to 7%. These findings suggest VTP-induced immune response can be augmented by prior immune modulation. Conclusions: Our results demonstrate that immunomodulation combined with local VTP enables systemic anti-tumor effects with impact on the development of early metastatic disease in the setting of poorly immunogenic tumors. The choice of different immune modulatory agents and timing of treatment appears to play a critical role in the treatment outcome. Further studies are in progress to evaluate combination of WST11-VTP with more than one immunomodulating agent to further characterize localized and systemic response and augment treatment success. These studies serve as pre-clinical support for clinical trials, exploiting the immunogenic effects of VTP for tumor ablation, being developed in collaboration with Memorial Sloan Kettering Cancer Center. Note: This abstract was not presented at the conference. Citation Format: Dina Preise, Natasha Kudinova, Uri Lindner, Keren Sasson, Ronny Uzana, Jonathan Coleman, Yoram Salomon, Avigdor Scherz. Immunomodulated VTP enables cure of metastatic prostate and breast cancers in animal models. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B012.