We present the results of combined fluorescence and optoacoustic monitoring of tumor treatment using novel photoactivatable multi-inhibitor liposomes with BPD and Irinotecan providing a synergetic effect of PDT and chemotherapeutic impact.
Genetically encoded photosensitizers are a unique instrument for investigation of cellular mechanisms of photodynamic therapy (PDT). Fluorescent flavoprotein miniSOG (mini Singlet Oxygen Generator) generates singlet oxygen with a high yield and demonstrates strong phototoxic properties in vitro in cancer cells. However, the effective approaches for PDT with miniSOG have not been developed so far. The purpose of the study is to investigate phototoxic effects of miniSOG induced by continuous wave (CW) or pulsed laser irradiation in tumor spheroids. We found that maximum photobleaching of miniSOG without temperature effects was achieved at 120 mW/cm2 in CW mode or pulse periodic mode. PDT in pulse periodic mode provided more pronounced increase in the number of dead cells in comparison with CW mode and, moreover, induced apoptosis more efficiently. Therefore, we report for the first time on an effective regimen for PDT with miniSOG in a tumor spheroid model using pulsed periodic laser irradiation.
A novel approach to monitor photosensitizer accumulation and photobleaching in the course of photodynamic therapy (PDT) with the use of nanoconstructs based on the simultaneous fluorescence (FL) and optoacoustic (OA) imaging is implemented. A liposome nanoconstructs employed in this studies contain benzoporphyrin derivatives (BPD) which serve as a photosensitizer and secondly, as a fluorophore, and the fluorescent IRDye800 dye acting as an additional contrasting agent due to its high quantum yield. FL provides visualization of BPD and IRDye800 distribution, while OA principle allows for BPD-absorption based imaging of tumor and its vascular environment. We demonstrate the results of a preliminary in vivo study with combined FL and OA custom-made setups on a NUDE mouse with human glioblastoma U- 87. The results of this studies show a hemorrhage in the tumor area on the OA images obtained @532 nm after PDT that is not visually detected, but confirmed with the following histological verification. Fast nanoconstructs accumulation (< 10 min) was observed using FL imaging with the concentration in tumor only 10% higher than in surrounding tissues. We believe that the ratio of nanoconstructs accumulation in tumor can be significantly increased using target approach.
To date, the effect of housing temperature on tumor development in the immunocompetent mice has been studied on poorly immunogenic cancer models. Standard housing temperature 20–26°C was shown to cause chronic metabolic cold stress and promote tumor progression via suppression of the antitumor immune response, whereas a thermoneutral temperature 30-31°C was more preferable for normal metabolism of mice and inhibited tumor growth. Our work represents the first attempt to discover the potential effect of housing temperature on the development of highly immunogenic tumor. EGFP-expressing murine colon carcinoma CT26 generated in Balb/c mice was used as a tumor model. No statistically significant differences were shown in tumor incidences and growth rates at 20°С, 25°С and 30°С for non-modified CT26. Maintaining mice challenged with CT26-EGFP cells at 30°С led to complete inhibition of tumor development. In summary, we demonstrated that the housing temperature is important for the regulation of growth of highly immunogenic tumors in mice through antitumor immunity.
The development of tumor therapies based on the activation of antitumor immunity requires tumor models that are highly immunogenic. The immunologic response to fluorescent proteins, green fluorescent protein (GFP), or enhanced GFP (EGFP) was demonstrated in different cancer models. However, for live animal imaging, red and far-red fluorescent proteins are preferable, but their immunogenicity has not been studied. We assessed the immunogenicity of the red fluorescent protein, KillerRed (KR), in CT26 murine colon carcinoma. We showed a slower growth and a lower tumor incidence of KR-expressing tumors in comparison with nonexpressing ones. We found that KR-expressing lung metastases and rechallenged tumors were not formed in mice that had been surgically cured of KR-expressing primary tumors. The effect of low-dose cyclophosphamide (CY) treatment was also tested, as this is known to activate antitumor immune responses. The low-dose CY therapy of CT26-KR tumors resulted in inhibition of tumor growth and improved mouse survival. In summary, we have established a highly immunogenic tumor model that could be valuable for investigations of the mechanisms of antitumor immunity and the development of new therapeutic approaches.
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