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Title:
Non-invasive blood flow monitoring using laser speckle contrast imaging of 4T1 murine tumor model after electroporation-based therapy
Authors:
Tadej Tomanica
Crt Kebera
Jost Stergara,b
Bostjan Markelcc,d
Tim Bozicc,e
Simona Kranjc Brezarc,e
Gregor Sersa*,c,d
Matija Milanica,b
aFaculty of Mathematics and Physics, Jadranska 19, 1000 Ljubljana, Slovenia
bJozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
cDepartment of Experimental Oncology, Institute of Oncology Ljubljana, 1000 Ljubljana, Slovenia
dFaculty of Health Sciences, University of Ljubljana, 1000 Ljubljana, Slovenia
eFaculty of Medicine, University of Ljublja
Abstract:
Monitoring tumor blood vessels during tumor growth, disease progression, and after treatment could provide valuable diagnostic information and advance knowledge about tumors and their microenvironment. Tumors require nutrients and oxygen for successful growth, and these needs are addressed by inducing angiogenesis – the formation of new blood vessels. However, newly developed blood vessels in tumors are generally disorganized, malformed, and leaky, which could lead to oxygen deprivation in diseased tissue. Abnormal vasculature affects the therapeutic success of various cancer treatments, such as radiotherapy and electrochemotherapy. The objective of this study was to monitor 4T1 murine tumor models grown subcutaneously in dorsal window chambers (DWC) non-invasively using laser speckle contrast imaging (LSCI). Specifically, we monitored the blood flow in blood vessels and tissue perfusion during tumor growth and after electroporation-based therapy.
LSCI is a real-time full-field non-invasive, contactless optical imaging technique broadly applied in biomedical applications to visualize blood flow. It is based on analyzing a speckle pattern arising from random interference of coherent laser light scattered by light-scattering particles such as red blood cells (RBCs). Particle movement leads to temporal intensity fluctuations, which results in speckle pattern blurring when recorded with a camera, and pattern blurring is related to the blood flow.
DWC was surgically implanted on the double skin layer on the back of 6 – 8 weeks-old female BALB/c mice. A ~10 mm diameter hole was cut in one layer of the skin, exposing the inner subcutaneous tissue of the other skin layer and associated vasculature. A cell suspension of 3 x 105 4T1 cells was injected into the remaining subcutaneous tissue. When the tumor volume reached 30 mm3, the treatment was carried out by intratumoral injection of 10 µg of plasmid DNA. Immediately after, gene electrotransfer was performed by application of 8 electrical pulses (1300 V/cm, 100 µs, 1 Hz) using plate electrodes with a 4 mm distance between the electrodes. During the experiments, mice were under 2 % (v/v) of isoflurane anesthesia.
The results show that blood flow in tumor vessels decreased by up to 70 % immediately after gene electrotransfer due to vasoconstriction following the electrical pulses. Moreover, on average, the overall tumor perfusion was 50 % lower than before the treatment. Four days after therapy, the blood flow and tumor perfusion did not change significantly for the treated tumors. However, there was a considerable increase (up to 10 times) in blood flow index (BFI) in a control tumor that did not receive any treatment, which could be attributed to a growing demand for nutrients and oxygen due to tumor growth.
In conclusion, we have shown that LSCI is sensitive to changes in blood flow in tumor vessels and tumor perfusion after electroporation-based therapy in real-time using a simple imaging technique based on the interaction between laser light and RBCs. Furthermore, we demonstrated that LSCI could estimate and visualize the blood flow in color-coded vascular maps, and thus we confirmed that LSCI could successfully monitor the vasculature of subcutaneously grown murine tumor models.
Keywords:
Refs:
Topic 1:
1. Biological responses (molecular, subcellular, cellular and intercellular)
Topic 2:
12. Biomedical applications
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