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1Cancer cells' response to chemotherapeutic treatment in the presence of collagen: monitoring with fluorescence and phosphorescence lifetime imaging techniques
I. Druzhkova1*, E. Nikonova23, A. Komarova14, A. Mozherov1, N. Ignatova1, I. Koryakina5, M. Zyuzin5, V. Baigildin6, Yu. Shakirova6, U. Lisitsa1, E. Shirshin23, V. Shcheslavskiy1, S. Tunik6,
M. Shirmanova1
1- Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University 2- Lomonosov Moscow State University, Faculty of Physics, Moscow, Russian Federation 3- Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University), Moscow, Russian Federation 4- Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod 5- School of Physics and Engineering, ITMO University, 9 Lomonosova St., St. Petersburg 191002, Russia 6- Saint-Petersburg State University, Institute of Chemistry, St. Petersburg, Russia
* danirin@yandex.ru
The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism, intracellular pH (pHi) as well as efficacy of chemotherapy [1]. Meanwhile, the role of collagen in differential cell response to treatment within heterogeneous tumor environment remains poorly investigated.
In this study, we compared effectiveness of chemotherapeutic drugs against cancer cells in vitro in the models with and without collagen. The effects of the drugs on cellular metabolism and intracellular pH (pHi) were investigated. For this, a combination of optical methods was used: Second Harmonic Generation (SHG) imaging of collagen, multiphoton fluorescence microscopy to visualize distribution of doxorubicine (DOX), fluorescence lifetime imaging (FLIM) of redox cofactor NAD(P)H [2], and phosphorescence lifetime imaging (PLIM) of pHi sensor. The pHi was followed using the new pH-sensitive probe BC-Ga-Ir [3].
We explored distribution of DOX in the presence of collagen with different structures in 3D collagen-based models and monitored metabolic changes in urinary bladder cancer cells using FLIM. Also, we monitored simultaneously the changes in pHi and metabolism in living colorectal cancer cells growing in collagen in vitro upon treatment with FOLFOX.
It was found that viability of different cancer cell types was better in the presence of collagen. Collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen [4]. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction of pHi) followed by a shift to more alkaline values. Notably, cancer cells changed cellular metabolism to a more oxidative state after treatment, but in the absence of collagen the changes were more pronounced [5].
Taken together, our data illustrate that tumor collagen contributes to heterogeneous and suboptimal response to investigated chemotherapeutic drugs and highlight the challenges in improving drug delivery and efficacy.
The study was supported by the Russian Science Foundation (project № 23-15-00294).
[1] M. Najafi, et al, Extracellular matrix (ECM) stiffness and degradation as cancer drivers, Journal of cellular biochemistry 120, 2782-2790 (2019).
[2] M. Shirmanova, et al, Exploring Tumor Metabolism with Time-Resolved Fluorescence Methods: From Single Cells to a Whole Tumor, Chapter 3 in Multimodal Optical Diagnostics of Cancer, pp. 133-155 (2020).
[3] J.R. Shakirova, et al, Intracellular pH Sensor Based on Heteroleptic Bis-Cyclometalated Iridium (III) Complex Embedded into Block-Copolymer Nanospecies: Application in Phosphorescence Lifetime Imaging Microscopy. Advanced Functional Materials 33, 2212390 (2023).
[4] I. Druzhkova, et al, Effect of Collagen Matrix on Doxorubicin Distribution and Cancer Cells' Response to Treatment in 3D Tumor Model, Cancers (Basel), Nov 8;14(22):5487 (2022).
[5] I. Druzhkova, et al, Monitoring the Intracellular pH and Metabolic State of Cancer Cells in Response to Chemotherapy Using a
Combination of Phosphorescence Lifetime Imaging Microscopy and Fluorescence Lifetime Imaging Microscopy, Int J Mol Sci. Dec 19; 25(1):49 (2023).
