Evaluation of CAR-T cell cytotoxicity against a prostate carcinoma model
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Evaluation of CAR-T cell cytotoxicity against a prostate carcinoma model

A.Kh. Valiullina a*, E.A. Zmievskaya a**, A.R. Rakhmatullina a***, M.N. Zhuravleva a****, E.E. Garanina a*****, R.R. Miftakhova a******, A.V. Petukhov b*******, A.A. Rizvanov a********, E.R. Bulatov a*********

aKazan Federal University, Kazan, 420008 Russia

bAlmazov National Medical Research Centre, Ministry of Health of the Russian Federation,

St. Petersburg, 197341 Russia

E-mail: *aigul1692@mail.ru, **ekazmievskaya@gmail.com, ***rahmatullina_2011@mail.ru, ****k.i.t.t.1807@gmail.com, *****kathryn.cherenkova@gmail.com, ******regina.miftakhova@gmail.com, *******alexeysakhalin@gmail.com, ********rizvanov@gmail.com, *********bulatovemil@gmail.com

Received December 4, 2021

 

ORIGINAL ARTICLE

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DOI: 10.26907/2542-064X.2022.2.212-230

For citation: Valiullina A.Kh., Zmievskaya E.A., Rakhmatullina A.R., Zhuravleva M.N., Garanina E.E., Miftakhova R.R., Petukhov A.V., Rizvanov A.A., Bulatov E.R. Evaluation of CAR-T cell cytotoxicity against a prostate carcinoma model. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2022, vol. 164, no. 2, pp. 212–230. doi: 10.26907/2542-064X.2022.2.212-230. (In Russian)

Abstract

In recent years, a new promising application of adoptive cell therapy (ACT) has been enabled by the development of technologies and the successful clinical use of T cells with a chimeric antigen receptor (CAR-T) for treating patients with malignant B-cell neoplasms. In this study, we obtained CAR-T cells with the second-generation chimeric antigen receptor FMC63-28Z-CAR. The effectiveness of the obtained CAR-T cells against the monolayer of the modified PC-3M prostate carcinoma cell line (Kat+CD19+) was evaluated. A correlation was demonstrated between an increased secretion of the proinflammatory cytokines IFNγ, TNFα, IL6 and a decreased confluence of the tumor cells monolayer. At the same time, a pronounced antitumor effect of CAR-T cells was observed starting from day 5 of coincubation. Thus, the proposed approach can potentially be applied for a preliminary evaluation of the effectiveness of biomedical CAR-T cell products in the treatment of solid tumors.

Keywords: CAR-T cells, chimeric antigen receptor, solid tumor, cell model, prostate carcinoma, PC-3M

Acknowledgments. This study was funded by the Russian Science Foundation (project no. 19-74-20026) and supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030). Part of the consumables (laboratory plasticware, interleukin-2) were purchased through a joint grant from the Foundation for Cancer Research Support (RakFond) and the SciStoreLab LLC.

Figure Captions

Fig. 1. Evaluation of the transduction efficiency of T lymphocytes by lentiviral particles. a – dot plot: the population of T cells on the forward and side scatter plot, the population of T cells is highlighted; b – dot plot: the population of GFP-positive T lymphocytes (CAR-T cells); c – dot plot: assessment of the CAR-T cells content by the signal level of biotinylated protein L conjugated with streptavidin-Pacific Blue; d – dot plot: the subpopulations of CD4+ and CD8+ in the total population of CAR-T cells. The upper left quadrant represents the population of CD4+ CAR-T cells, the lower right quadrant represents the population of CD8+ CAR-T cells.

Fig. 2. Evaluation of the transduction efficiency of the PC-3M cell line with lentiviral particles encoding for the red fluorescent protein Katushka2S and CD19 antigen. a – dot plot: the unmodified control line PC-3M; b – dot plot: the level of transduction of PC-3M cells by lentiviral particles encoding for the red fluorescent protein Katushka2S; c – dot plot: the unmodified control line PC-3M; d – dot plot: the level of transduction of PC-3M(Kat+) cells by lentiviral particles encoding for CD19 antigen.

Fig. 3. Evaluation of the CAR-T cells effectiveness against the tumor cell line PC-3M(Kat+CD19+) in the dynamic evaluation of the changes in confluence from days 1 to 7 of co-cultivation. The results are presented as the mean value, the error bars indicate the standard deviation (n = 3, p < 0.05).

Fig. 4. Micrographs of the monolayer of the tumor cell lines PC-3M(Kat+) and PC-3M(Kat+CD19+) co-cultivated with CAR-T cells. Light microscopy, fluorescence microscopy, representative micrographs (n = 6) are shown, scale bar is 100 µm. CAR-T cells – green fluorescence. Tumor cells PC-3M(Kat+) and PC-3M(Kat+CD19+) – red fluorescence.

Fig. 5. Heat map of the levels of expression of IFNγ, IL6, and TNFα in the culture medium during the co-cultivation of CAR-T cells with the tumor cell lines PC-3M(Kat+) and PC-3M(Kat+CD19+) at different time points.

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