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www.nature.com/cddiscovery ABSTRACTS COLLECTION OPEN “Recent Advances in Cancer Research” Bern, November 4-5, Cell Death Discovery (2022) 8:1–11; https://doi.org/10.1038/s41420-022-01249-1 Sponsorship: Publication of this supplement was funded by Nanotech Innovations LTD, who collected and prepared the abstracts for publication. All content was reviewed and approved by the Scientiﬁc Committee at Kazan Federal University, which held full responsibility for the abstract selections. Webpage for the event: http://cancerproteostasis.com The role of autophagy in Hashimoto’s thyroiditis rapamycin, mTOR in patients with HT is signiﬁcantly higher than in T cells of healthy control group. Compared with the group of healthy donors, the levels of both autophagy initiator Anastasia Burtseva, Zinaida Abramova proteins (ATG14 and Vps34), and elongation proteins (LC3-I and LC3-II) are also higher in T cells of patients with HT. Laboratory of Immunopathology, Kazan Federal University, Kazan, The analysis showed a correlation between the levels of Russia; ORCID: 0000-0003-3749-3411 LC3-I and LC3-II proteins and the functional state of the thyroid gland. Introduction: Hashimoto’s thyroiditis (HT) is currently the most Conclusion: We demonstrated that mTOR activity is increased common autoimmune disease in humans, and the most in T-cell from HT patients. These results suggest that abnormally common cause of hypothyroidism (occurring in approximately activated mTOR activity could may be responsible for impaired 5% of the population). Autophagy is a multifactorial regulated autophagy, regulate the survival of autoreactive T cell during HT, cell death mechanism that may play a dual role in different and could thus lead to design new therapeutic options for thyroid diseases or at different stages of the same disease. The treatment Hashimoto’s thyroiditis. recent studies have conﬁrmed that activation of both autophagy Funding: This study was part of the Kazan Federal University itself and various disorders in the autophagic pathway can Strategic Academic Leadership Program (PRIORITY-2030). mediate inﬂammatory responses and immune hyperactivation, Disclosure: The authors declare no competing interests. which is closely related to the pathogenesis of autoimmune diseases, including HT. This study has shown that the mTOR signaling pathway in Evaluation of immune cell cytotoxic activity after interaction T cells is closely associated with autophagy abnormalities and may with tumour-derived membrane vesicles play an important role in the pathogenesis of HT. Materials and methods: To evaluate autophagic activity in Ivan Filin, Kristina Kitaeva, Anna Gorodilova, Yuri Mayasin, isolated peripheral T lymphocytes, we examined these cells for Chulpan Kharisova, Daria Chulpanova, Valeria Solovyeva, the presence of autophagosomes by transmission microscopy, Albert Rizvanov ﬂow cytometry, and Western blot for autophagosomal markers. Peripheral blood mononuclear cells (PBMCs) were isolated by Institute of Fundamental Medicine and Biology, Kazan Federal Ficoll-Hypaque density-gradient centrifugation. CD3 T cells University, Kazan, Russia; ORCID: 0000-0002-3661-0527 were isolated using negative selection (DynaBeads Untouched T-cell Isolation Kit, Invitrogen), with routine purity >95%. The HT group consisted of 20 HT patients. Clinical diagnosis of HT Introduction: Cytochalasin B-induced membrane vesicles (CIMVs) was based on elevated serum levels of thyroid antibodies are membrane-bound structures of various sizes, that contain (TPOAb> 200 IU/ml). The healthy control (HC) group consisted of proteins, lipids, nuclear and mitochondrial components. CIMVs are 29 participants. able to fuse with recipient cells via endocytosis. Therefore, CIMVs Results: Autophagic vacuoles quantiﬁed by electron micro- from tumor cells can be used to present antigens to cells of the scopy are also found to be signiﬁcantly more frequent in T cells immune system. from HT patients compared with healthy controls. This Materials and methods: CIMVs were obtained from human elevated number of autophagic structures is not distributed melanoma M-14 cells using cytochalasin B. Peripheral blood homogeneously and appears to be more pronounced in certain monocytes (PBMCs) were isolated by Ficoll gradient centrifugation T cells. Also, lipofuscin granules and multilayered autophago- (1,077 g/cm ). Differentiation of dendritic cells (DCs) from PBMCs was somes were found in T lymphocytes of HT patients. Levels of reached by cultivation of PBMCs with a cocktail of cytokines for negative regulator of autophagy, the mammalian target of 7 days. Co-cultivation of DCs and CIMVs was carried out for 48 h, and Ofﬁcial journal of CDDpress 1234567890();,: 2 then PBMCs were added. Afterward PBMCs were co-cultivated with Tamoxifen suppresses NLRP3 priming via miR-223 in breast M-14 cells for 24 h and then M-14 cells were stained with the cancer independently from ERα antibodies containing a ﬂuorescent label for annexin V. The results 1 1 were analyzed by ﬂow cytometry. Shaimaa Hamza , Ekaterina E. Garanina , Svetlana F. Results: We analyzed the cytotoxicity activity of activated 1 2* Khaiboullina , Gulcin Tezcan PBMCs after interaction with mature DCs using confocal micro- scopy. It was shown that the number of apoptosis M-14 cells Institute of Fundamental Medicine and Biology, Kazan Federal increased in 20% after cultivation with activated PBMCs than after University, 420008 Kazan, Russia; Department of Fundamental cultivation with non-activated PBMCs, and in 23% compared to Sciences, Faculty of Dentistry, Bursa Uludag University, Bursa 16059, control M-14 cells. Turkey; ORCID ID: 0000-0002-5012-4760 Conclusions: Thus, due to the ability of CIMVs to present tumor antigens to DCs and activate the antitumour immune response, Introduction: Tamoxifen is drug used for breast cancer CIMVs of tumor cells are a promising object for the development chemotherapy. However, the resistance to tamoxifen was of therapeutic antitumour vaccines. However, further studies are demonstrated in some cases. The mechanisms of this resistance needed in this area to study possible ways of modulating the remain largely unknown, while the inﬂammation was suggested immune response. as playing an important role. Inﬂammation could be mediated by Funding: This study was part of the Kazan Federal University Nod-like receptor protein 3 (NLRP3) inﬂammasome, which was Strategic Academic Leadership Program (PRIORITY-2030) shown to promote proliferation, survival, metastasis, angiogenesis, andfundedbythe RussianScience Foundation grant21-74- and immunosuppression of breast cancer cells. The NLRP3 priming step could be regulated by post-transcriptional modiﬁca- Disclosure: The authors declare no competing interests. tion using microRNA (miRNA). MiR-223, a miRNA with tumor suppressing capacity, was shown to block translation of NLRP3 mRNA and suppress the breast cancer cell growth. The aim of the study: In this study, NLRP3 inﬂammasome Functional characterization of the interaction between TG2 activation in breast cancers cell lines, MCF-7 (ER-α ) and, MDA- with p53 tumor suppressor protein in lung adenocarcinoma MB-231(ER-α ), was analyzed. Also, the effect of tamoxifen on cells regulation of miR-223/NLRP3 axis in these cell lines was 1 1 1,2 investigated. Yuliya Gnennaya , Evgenii Smirnov , Nikolai Barlev Materials and methods: Lipopolysaccharide (LPS)/ATP was used to activate NLRP3. Glybenclamide/LPS served as a control for Laboratory of Regulation of Gene Expression, Saint-Petersburg, NLRP3 suppression. Western blot was used to conﬁrm NLRP3 Russia; ORCID: 0000-0002-3571-333X activation and Gasdermin D (GSDMD) cleavage, while IL-1β secretion was shown by ELISA. Apoptosis and proliferation kinetics Introduction: Transglutaminase type 2 (TG2) is a multifunctional were analyzed by immune blotting of BAX protein, Annexin V and enzyme that mediates a wide range of covalent modiﬁcations of cell proliferation assay. Statistical analysis was done using one-way proteins. TG2 is involved in pathogenesis of a number of neoplastic ANOVA with Tukey’s analyses, Kruskal–Wallis one-way analysis of and ﬁbroproliferative diseases, including malignant tumors and variance, independent sample t-test and linear regression analysis. ﬁbrosis of the lungs, kidneys, and heart. High levels of TG2 in cells Results: LPS/Tamoxifen suppressed NLRP3 transcription, of several types of lung cancer and RCC (renal cell carcinoma) induced miR-223 expression and IL-1β secretion as compared to correlate with poor patient survival and increased resistance to LPS-only in both cell lines (p < 0.001). Additionally, NLRP3 chemotherapy drugs. Apart from the fact that TG2 inhibition activation reduced the tumor cell growth in both cells lines, suppresses the RCC tumor growth by p53-mediated apoptosis, little whereas suppressing NLRP3 could result in tumor cell line speciﬁc detail is known about the role of TG2 in modulating p53 activity in the growing pattern. context of lung cancer. Conclusions: Our ﬁndings suggest that tamoxifen could Materials and methods: We created a number of isogenic suppress the expression of NLRP3 by inducing miR-223 expression adenocarcinoma cell lines with different p53 status (wt vs mut) and in breast cancer cells. However, the tamoxifen mediated suppres- explored the potential mechanism of TG2-mediated effects on the sion of NLRP3 increased the proliferation of tumor cells lacking intracellular distribution of p53 and subsequently, its functions. We ER-α, whilst it attenuated proliferation of ER-α-expressing breast validated the ability of these proteins to interact using GST pull-down cancer cells. and CoIP assay. Furthermore, WB and qPCR, to analyze the alterations Funding: This work was supported by Russian Science in the levels of p53-dependent proteins. To evaluate the effect of TG2 Foundation grant #21-74-00048. on p53-mediated sensitization and cell death of the obtained cell lines Disclosure: The authors declare no competing interests. upon treatment with etoposide combined with clinically approved TG2 inhibitor cysteamine, we performed a battery of assays, including cell cycle analysis, colony formation test, xCELLigence proliferation Mesenchymal stem cells derived microvesicles suppress analysis, etc. activation of T and B-lymphocytes Results: Together, our data strongly suggest that TG2 down- regulates the expression and activity of wild-type p53 but not its 1 1 Sirina Kurbangaleeva , Sevindzh Kletukhina , Marina mutant form. This effect was abrogated by blocking the cross- linking activity of TG2 by cysteamine, which results in the Gomzikova augmented stability of the p53 protein. Conclusion: Collectively, TG2 can be considered as an Laboratory of Intercellular communication, Kazan Federal Univer- oncogene that inactivates p53 response to DNA damage. sity, Kazan, 420008, Russia; ORCID: 0000-0003-1637-1888 Funding: The work was supported by the grant from RSF # 20- 15-00189. Introduction: Mesenchymal stem cells (MSCs) derived extracel- Disclosure: The authors declare no competing interests. lular vesicles (EVs) demonstrate immunosuppressive effects on Cell Death Discovery (2022) 8:1 – 11 3 ΔM T cells, B-cells, dendritic cells, and macrophages. However, EVs is Results: Opa1 macrophages could not stimulate the pro- difﬁcult to obtain in the desired amounts, so we used cytochalasin liferation of cancer cells. This phenotype does not depend on B to mass-produce microvesicles. The aim of our work was to phagocytic abilities of macrophages, but is strongly associated evaluate the immunosuppressive activity of induced microvesicles with the secretome of macrophages and their failure in re- (iMV) of MSCs using model of peripheral blood mononuclear cells programming into the pro-tumorigenic M2-like subtype. (PBMCs) activation using phytohemagglutinin (PHA) in vitro. Conclusions: Opa1 regulates activation of TAMs, leading to the Material and methods: Treatment of MSCs with cytochalasin B production of secreted factors required for tumor growth. Drugs (10µg/ml) and vortexing induce iMV-MSCs formation in a large inducing mitochondrial dysfunction in macrophages might be scale. PBMCs were pre-treated with iMV-MSCs for 24 h followed by prominent candidates for anticancer therapy. incubation with PHA (10 µg/mL) for 72h. The percent of activated Funding: This work was supported by the grant from Russian CD25 expressing T-helpers (CD4+CD25+), B-cells (CD19+CD25+), Government No. 075-15-2021-600. and T-cytotoxic lymphocytes (CD8+CD25+) in the PBMCs was Disclosure: The authors declare no competing interests. analyzed by ﬂow cytometry. Results: We found that iMVs themselves do not inﬂuence on activation status of PBMCs, since no differences between expression of CD25+ in control PBMCs and iMV-MSCs treated Methyltransferase Set7/9 regulates sensitivity of HER2- PBMCs were detected. Treatment with PHA-induced activation in positive breast cancer cells to the genotoxic stress T-helpers by 34.64 times (p = 0.000005), in B-cells by 3.12 times (p = 0.0002), and in T-cytotoxic lymphocytes by 87.36 times Dmitry Myadelets, Sergey Parfenyev, Julia Vasileva, Oleg (p = 0.000001) compared with control. Conversely, pre-treatment Semenov, Olga Fedorova, Oleg Shuvalov, Alexey Petukhov, of PBMCs with iMV-MSCs decreased subsequent PHA-induced Alexandra Daks* activation of PBMCs by 2.3 times in T-helpers (0.0006), by 2.02 times in B-cells (0.0004), and by 4.38 times in T-cytotoxic cells Laboratory of Gene expression Regulation, Institute of Cytology RAS, (0.000005). St Petersburg, Russia; *ORCID: 0000-0003-0495-1244 Conclusion: We found that iMV-MSCs show signiﬁcant suppres- sion on phytohemagglutinin-induced activation of PBMCs in vitro. Introduction: HER2-positive phenotype of breast cancer (BC) is Funding: This work was funded by RSF research project №21- considered to be one of the most aggressive form of this disease 75-10035 and supported by the Kazan Federal University Strategic due to high intensity of tumor growth and metastasis formation. Academic Leadership Program (PRIORITY-2030). Elevated expression of HER2 is an unfavorable prognostic marker Disclosure: The authors declare no competing interests. for BC patients. Additionally, it was repeatedly shown that HER2- positive BC cells are characterized by the ability to acquire the resistance to genotoxic therapy. Set7/9 is a methyltransferase that modiﬁes a list of histones and Targeting mitochondrial functions in tumor-associated non-histone targets with lysine residues thus affecting their macrophages as a strategy for cancer therapy functions, stability and cellular localization. Set7/9 was shown to methylate cancer-associated transcription factors including p53, 1 1 Adelya Mullakhmetova , Yana Mukhamedshina , Marina E2F1, estrogen receptor α (ERα), NFkB, STAT3, YAP, androgen 1 1 1 2 Gomzikova , Albert Rizvanov , Nick Barlev , Nikita Markov , receptor (AR) and others. 1,2 3 Hans-Uwe Simon , Anna Brichkina In this study we investigated the role of Set7/9 in genotoxic stress response and cisplatin resistance of HER2-positive human BC cells SKBR3. Laboratory of Molecular Immunology, Institute of Fundamental Materials and methods: Set7/9 knockdown was performed by Medicine and Biology, Kazan Federal University, Kazan, Russia; lentiviral transduction of BC cells with subsequent puromycin Institute of Pharmacology, University of Bern, Bern, Switzerland; selection. The cytotoxic effect of cisplatin was investigated using Center for Tumor and Immune Biology, Clinic of Gastroenterology, MTT test and by registration of cell index via xCelligence cell Endocrinology, Metabolism and Infectiology, Philipps University of analyzer. The apoptosis level was assessed using annexin V/PI Marburg, Marburg, Germany; ORCID: 0000-0001-8707-9900 double staining and ﬂow cytometry analysis. The expression of excision repair factors was determined both by qPCR and western Introduction: Tumor-associated macrophages (TAMs) represent blot analysis. a major population of immune cells inﬁltrating solid tumors Results: We revealed that knockdown of Set7/9 in SKBR3 cells and are often associated with a poor patient prognosis. led to acquisition of resistance to cisplatin and to decrease of Pro-tumorigenic M2-like macrophages tend to exert immune- apoptosis level under cisplatin treatment. We also demonstrated suppressive effects, favouring tumor progression. Pro- that Set7/9 suppression causes activation of such DNA excision tumorigenic functions of TAMs require massive metabolic repair factors as ATR, Fen1, Rad51, MPG, APE1, XRCC1 both at rearrangements linked to mitochondria and rely on intact mRNA and protein levels. mitochondrial respiration. Therefore, targeting the mitochondrial Conclusions: According to our data, methyltransferase Set7/9 metabolism appears to be a promising strategy to diminish the affects the sensitivity of HER2-positive BC cells to cisplatin pro-tumorigenic effect of macrophages. treatment via regulation of excision repair factors under genotoxic Materials and methods: We utilized a mouse model with ΔM conditions. These ﬁndings contribute to investigation of drug conditional deletion of Opa1 in macrophages (Opa1 )to resistance acquisition mechanisms and allow to consider Set7/9 induce mitochondrial dysfunction. We cultivated tumor cells expression as a biomarker of genotoxic therapy response of HER2- with bone marrow-derived macrophages of wild-type or positive BC. Opa1-/- or their conditioned media. The activation status of Funding: This work was supported by the grant from RSF#19- macrophages was evaluated by stimulation with IL4 into M1- 75-10059. subtype or IFNγ+LPS into M2 followed by qPCR analysis of Disclosure: The authors declare no competing interests. marker genes. Cell Death Discovery (2022) 8:1 – 11 4 Evaluation of viability, cytokine proﬁle changes and Zeb1 and Zeb1-controlled cellular processes are tightly associated pluripotency genes of colorectal cancer spheroids cells after with formation and progression of different cancer types including interaction with tumor and stem vesicles breast and lung cancers. Methyltransferase Set7/9 acts as a transcription regulator due to 1 1 1 its ability to methylate histones H3, H1, H1.4, H2A and H2B thus Aleksei Ponomarev , Anna Kurnenkova , Zarema Gilazieva , affecting the genes expression. Additionally, Set7/9 was shown to 1 1 Albert Rizvanov , Valeriya Solovyeva methylate different transcription factors e.g. p53, E2F1 and NFkB and as a consequence to affect gene expression indirectly. In this Laboratory of Gene and cell technology, Institute of Fundamental study we focused on Set7/9 effect on Zeb1 expression and the Medicine and Biology, Kazan Federal University, Kazan, Russia; role of Set7/9 in regulation of migration potential of cancer cells. ORCID: 0000-0003-2320-6119 Materials and methods: H1299 and A549 human lung cancer cells and MDA231 human breast cancer cell line were used in this Introduction: Extracellular vesicles are membrane structures that study. The migration activity of cancer cells with different Set7/ have a relevant role in intercellular communication because they 9 status was investigated using scratch test and cell index have the ability to transport lipids, transcription factors, mRNA, registration (xCelligence cell analyzer). The levels of Zeb1 and and proteins. There is evidence of a special role of vesicles in markers of epithelial and mesenchymal phenotypes were studied cancer progression. Therefore, the study of the vesicle effect on both by qPCR and western blot analysis. tumor spheroids is important. The aim of this work is to study the Results: As a result of this study, we showed that Set7/ effect of cytochalasin B-induced membrane vesicles (CIMVs) of 9 silencing led to activation of migration potential, while Set7/9 colorectal cancer spheroids in vitro. overexpression on the contrary suppressed the motility of tested Materials and methods: In this study, сolorectal cancer cell line cell lines. We also demonstrated that Set7/9 regulates Zeb1 and (HCT-15) was used to create tumor spheroids. Glioblastoma cell E-cadherin expression, that elucidates the mechanism of the line (SNB-19) and adipose derived mesenchymal stem cells (MSCs) observed effect. Thus, we revealed the new mechanism regulating were used to isolate MVs. Vesicles from SNB-19 (SNB-19 CIMVs) cancer cell migration ability through Set7/9-Zeb1-E-cadherin axis. and MSCs (MSC CIMVs) were isolated using 10 μg/ml of Conclusions: We revealed that methyltransferase Set7/9 affects cytochalasine B and a series of sequential centrifugations. The the migration potential of cancer cells and Zeb1 expression. Since addition of SNB-19 CIMVs and MSC CIMVs to spheroids was carried metastasis formation remains a scourge of oncology, this study out at concentrations of 1, 2 and 5 μg. The effect of SNB-19 CIMVs may contribute to the development of the effective strategies of and MSC CIMVs was analyzed using ﬂow cytometry, multiplex anticancer therapy. analysis, and real-time PCR. Funding: This work was supported by the grant from RSF# Results: After addition of MSC CIMVs, there was no signiﬁcant 19-75-10059. change in cell viability in the spheroid. However, addition of SNB- Disclosure: The authors declare no competing interests. 19 CIMVs to colonosphere, there was a dose-dependent increase cell viability. OCT4 and Nanog mRNA levels were increased after addition of SNB-19 CIMVs but decreased after MSC CIMVs. Evaluation of microvesicles stability using ﬂow cytometry Cytokine analysis showed signiﬁcant differences in 40 major approach cytokines in colonospheres with SNB-19 CIMVs and MSC CIMVs. Conclusions: Thus, it was shown the possible effect of SNB-19 1 1 Sevindzh Kletukhina , Marina Gomzikova CIMVs and MSC CIMVs on tumor spheroids. Further research of this mechanism effects is necessary. Laboratory of Intercellular communication, Kazan Federal Univer- Funding: This study was part of the Kazan Federal University sity, Kazan, 420008, Russia; ORCID: 0000-0003-1637-1888 Strategic Academic Leadership Program (PRIORITY-2030) and funded by the Russian Science Foundation grant 21-74-10021. Disclosure: The authors declare no competing interests. Introduction: Microvesicles (MVs) are spherical membrane structures released from the cell surface and ranging in size from 50 to 2000 nm. Today, the use of MSCs derived MVs in medicine as therapeutic agents and drug delivery vectors is of great interest. Downregulation of Set7/9 increases migration activity of Therefore, it is important to develop high throughput methods for cancer cells through Zeb1 expression activation assessing the yield and integrity of isolated or stored MVs. The aim of our work was to develop a method for assessing the yield and Oleg Semenov, Sergey Parfenyev, Olga Fedorova, Oleg integrity of MVs using the ﬂow cytometry approach. Shuvalov, Alexey Petukhov, Alexandra Daks* Materials and methods: MVs were obtained from MSCs using 10 µg/ml cytochalasin B, which is an agent for the cell Laboratory of Gene expression Regulation, Institute of Cytology RAS, cytoskeleton disorganization and mass production of MVs. MVs St Petersburg, Russia; *ORCID: 0000-0003-0495-1244 were loaded with CFDA SE dye to monitor their integrity. MVs were resuspended in saline and stored at 4°C, -20°C or at 25°C, Introduction: Migration ability is one of the key characteristics of freeze dried and stored at -20°C. Quantity and integrity of MVs cancer cells aggressiveness due to its contribution to metastasis were analyzed using ﬂow cytometry with enhanced detector (BD formation. Due to epithelial-to-mesenchymal transition process FACS Aria III BD Bioscience, USA). (EMT) the epithelial cells attached to basement membrane acquire Results: We found that storage of MVs in saline at 25°C for the ability to migrate into the blood vessels with subsequent 28 days led to decrease of MVs amount by 43%. Whereas the formation of new cancer foci. Zeb1 is one of the key EMT- integrity of MVs was preserved at 64%. Lowering the storage orchestrating transcription factors that suppresses the expression temperature to 4°C allows to extend the shelf life of MVs in 4 times of epithelial markers such as E-cadherin, cytokeratins and integrins to 112 days with the same MVs parameters. Next, we evaluated and contributes to stabilization of mesenchymal factors e.g. the stability of MVs suspended in saline and stored at –20°C. After vimentin and N-cadherin. In addition to EMT Zeb1 was shown to 112 days of storage, the total number of MVs decreased by 20.5%, contribute to other cancer-associated characteristics such as drug while the integrity was preserved at 71%. Widely used method for resistance, stemness and cellular senescence. Dysregulation of long-term storage of bioactive drugs is freeze-drying. We found Cell Death Discovery (2022) 8:1 – 11 5 that freeze-drying and storage of MVs during 112 days at –20°C Characterization of microglia activation in a model of spinal led to reduction of MVs amount by 51%, with the percent integrity cord injury different severity in vitro of the MVs being estimated at 86%. 1 1 Conclusion: Thus, evaluation of MVs amount and integrity Elvira Akhmetzyanova , Anna Timofeeva , Yana using the ﬂow cytometry method demonstrated that freezing and Mukhamedshina storage at –20°C is the most suitable option for storing MVs in solution. This allows to maintain a high percentage of integrity Kazan Federal University, Kazan, Russia; ORCID:0000-0002-5751- and high delivery efﬁciency by MVs. Funding: This work was funded by RSF research project №21- 75-10035 and supported by the Kazan Federal University Strategic Introduction: Inﬂammatory reactions that occur after spinal Academic Leadership Program (PRIORITY-2030). cord injury (SCI) are a protective mechanism of the organism Disclosure: The authors declare no competing interests. aimed at minimizing the consequences of injury. However, these mechanisms do not always aim to the preservation of the neural tissue functionality. It is considered that the acquisition of a certain phenotype by microglia/macrophages is one of the key Interleukin-8 overexpression in tumor modulates progression processes that determine the outcome of post-traumatic in colorectal cancer reactions in the spinal cord. Here we studied microglia activation and M1/M1 polarization in a model of mild, moderate 1,2 1 1,3 Irina Bogomolova , Dinara Dolgova , Inna Antoneeva , and severe SCI in vitro. 1 1 1 Ilseya Myagdieva , Tatyana Abakumova , Tatyana Gening Materials and methods: SCI different [mild (1.5 m/s, n = 20), moderate (2.5 m/s, n = 20) and severe (4 m/s, n = 20)] severity was 1 2 Ulyanovsk State University, Ulyanovsk, Russia; Federal Scientiﬁc induced by an impactor (Impact One ™ Stereotaxic Impactor, and Clinical Center for Medical Radiology and Oncology Federal Leica) at Th8 level. After 3, 7, 14, and 60 days post injury (dpi) Medical and Biological Agency of Russia, Dimitrovgrad, Russia; experimental and intact rats were anesthetized with overdosed Regional Clinical Oncologic Center, Ulyanovsk, Russia; ORCID: 0000- isoﬂurane anesthesia and intramuscular injection of Zoletil (20mg/ 0003-3331-8632 kg, Virbac Sante Animale). Then a fragment of the spinal cord obtained from the site of injury/Th8 was prepared and homo- genized. To modeling the SCI in vitro, spinal cord extracts were Introduction: Currently, a search is underway for speciﬁc added at 4-5 days cultivation of microglia isolated from neonatal molecular markers for predicting the sensitivity of colon tumors rat cerebral cortex. To analyze cells by ﬂow cytometry, we used to chemotherapy. Interleukin-8 (CXCL8) is considered as a markers for CD86, CD45, and CD206. To characterize the cytokine promising pro-inﬂammatory marker of carcinogenesis associated proﬁle of microglia supernatants cultivated under different with tumor angiogenesis, invasion through activation of the conditions, multiplex analysis was performed using the Bio-Plex epithelial–mesenchymal transition, secondary neutrophil chemo- Pro Rat Cytokine 23-Plex Immunoassay (Bio-Rad). taxis into the tumor zone. The aim of this work is to evaluate the Results: It was shown, microglia have a consistently high level level of CXCL8 in the tumor as a marker of progression in of CD86 expression in the ﬁrst week of cultivation. The expression colorectal cancer (CRC). level of CD45 under different conditions of microglia cultivation Materials and methods: We isolated RNA using Sileks-MagNa ranged from 40 to 60%. The expression of CD206 had the lowest magnetic particles (Sileks, Russia) in 49 FFPE tumor samples from values in a model of mild and moderate SCI at 14 and 60 dpi. patients with stage II-III CRC. Next, we performed quantitative RT- According to the results of multiplex analysis, signiﬁcant changes PCR using Sybr Green dye. When calculating the normalized in the concentration of TNF-α, IFN-γ and MCP-1 between the expression of CXCL8 (ΔΔСq), the GAPDH gene was used as a studied groups were found. It is noteworthy that the expression of referee gene. The Kaplan-Meier test (STATISTICA 13.0 (StatSoft, all studied cytokines was consistently high when microglia were USA) was used to assess the effect of the level of CXCL8 in the cultivated with an intact spinal cord. tumor on progression-free time. Conclusion: It was shown that the severity of SCI has a decisive Results: We have found that the expression of CXCL8 in cells of inﬂuence on microglia activation and M1/M2 polarization adenocarcinoma of the intestine with low differentiation (Med proceeds equally actively in all models of SCI in vitro. Multiplex 8.770 (1.127–15.114) is statistically signiﬁcantly higher than in the analysis and ﬂow cytometry data demonstrated activation of both group with moderate and high differentiation (p1 = 0.004; M1 and M2 microglia in models of mild, moderate and severe SCI. p2 = 0.012). When comparing groups with different responses to Funding: This study was supported by the Russian Foundation chemotherapy, it was found that in the group of patients with for Basic Research (to YM) (Grant No. 20-34-90060) and was part of disease progression (against the background of chemotherapy), the Kazan Federal University Strategic Academic Leadership the level of CXCL8 mRNA in the tumor was statistically signiﬁcantly Program (PRIORITY-2030). higher compared to patients with early relapse and without Disclosure: The authors declare no competing interests. relapse for more than 3 years (p1 = 0.0008; p2 = 0.001). Thus, an increase in CXCL8 expression in the primary tumor increases the relative risk of CRC progression after standard chemotherapy. Dependence on the age of patients, the stage of Recognition of cysteine mutant forms of the sodium- the disease, the presence of mutations in the EGFR-signaling dependent phosphate transporter NaPi2b by monoclonal pathway on the level of CXCL8 in the tumor was not revealed. antibodies in ovarian cancer cells OVCAR-8 Probably, the effect of CXCL8 on progression is mediated through modulation of the inﬂammatory response in the microenviron- 1 1 1 Leisan Bulatova , Daria Savenkova , Daria Reshetnikova , ment, activation of tumor stem cells. 1 1 1 Arina Timonina , Alsina Nurgalieva , Vera Skripova , Mikhail Conclusions: Tumor overexpression of CXCL8 is associated with 2 1 short progression-free time in CRC patients after standard Bogdanov , Ramziya Kiyamova FOLFOX/HELOX therapy and can be considered as an indepen- dent marker of progression. Research laboratory Biomarker, Institute of Fundamental Medicine Disclosure: The authors declare no competing interests. and Biology, Kazan Federal University, Kazan, Russia; McGovern Cell Death Discovery (2022) 8:1 – 11 6 Medical School at The University of Texas Health Science Center at in high-penetrance genes (CDH1, MLH1, MSH2, TP53) and 143 Houston, Houston, USA; ORCID: 0000-0001-6696-8477 (37.5%) genetic alterations in the genes with mild to low penetrance. Among the interesting ﬁndings, we would like to highlight a breast cancer case with a c.-39-1_-39del variant located Introduction: The sodium-dependent phosphate transporter outside the 5’UTR region of BRCA2 gene, which is rarely included NaPi2b is a target for monoclonal antibody (mAb) therapy since in gene panels. it’s overexpressed in several malignancies. The extracellular Conclusion: Multigene panel testing allows for a differential domain (ECD) of the NaPi2b (188-361 a.a.) contains 4 cysteines diagnosis and identiﬁcation of a high-risk group for oncological and the MX35 epitope (324-338 a.a.). It is known that DTT diseases. The inclusion of non-coding gene regions into HCPS reduces disulﬁde bonds, limiting MX35 recognition by mAbs in gene panels is highly important for identiﬁcation of rare Western blot (WB) analysis. We suggest that cysteines around the spliceogenic variants with high penetrance. MX35 epitope form disulﬁde bonds, altering the NaPi2b ECD Disclosure: The authors declare no competing interests. conformation. Materials and methods: NaPi2b mutants with cysteine-to- alanine substitutions at positions 303, 322, 328 and 350 were 3+ 3+ generated. The mutant forms were transfected into OVCAR-8 Ce ,Tb :YF nanoparticle-polymer -“radaclorin” conjugates ovarian cancer cells and analyzed with mAbs using Western blot for X-ray induced photodynamic therapy and ﬂow cytometry. Results: OVCAR-8 cells expressing wild-type NaPi2b showed a 1 Alexey S. Nizamutdinov, Elena V. Lukinova , Nail speciﬁc signal only without DTT as usual, while all NaPi2b cysteine 2 2 1 Shamsutdinov , Pavel V. Zelenikhin , Alina Khusainova , mutants showed no signal in WB even without DTT. According to 1 Maxim S. Pudovkin the results of ﬂow cytometry, the proportion of MX35-positive OVCAR-8 cells that produce cysteine mutants of NaPi2b is Institute of physics, Kazan Federal University, Kazan, Russia, signiﬁcantly lower compared to the wild type of the transporter. Institute of Fundamental Medicine and Biology, Kazan Federal Conclusions: Cysteines C303, C322, C328, and C350 are University, Kazan, Russia; ORCID: 0000-0001-8707-9900 implicated in the creation of disulﬁde bonds within ECD of NaPi2b. This is an essential step in the production of the NaPi2b Introduction: Photodynamic therapy (PDT) is a modern and non- ECD conformation, which is necessary for the recognition of the invasive form of therapy, used in the treatment of cancers and MX35 epitope by mAbs. non-oncological diseases. It is based on the use of photosensiti- Funding: This paper has been supported by the Kazan Federal zers, which are accumulated in pathological tissues. The modern University Strategic Academic Leadership Program (PRIORITY- photosensitizers are activated by visible light which cannot 2030) and partly supported by by Russian Science Foundation penetrate the biological tissues deeper than 1 cm. Hence, the Grant RCF 20-14-00166. conventional PDT can be used only for superﬁcial diseases. Disclosure: The authors declare no competing interests. There is an approach allowing using X-ray irradiation in order to overcome these limitations by creating conjugates of nanoparti- cles and photosensitizers. Thus, the main objectives of the study Application of multigene panels in hereditary cancer 3+ 3+ were to create the Ce ,Tb :YF nanoparticles-“radaclorin” predisposition testing conjugates and to study biological activity (cytotoxicity and cellular uptake). 1 1 1,2 Sergey Nikolaev , Anastasia Danishevich , Airat Bilyalov , 3+ 3+ Materials and methods: Ce ,Tb :YF nanoparticles were 1 1 2,3,4 3 Natalia Bodunova , Igor Khatkov , Oleg Gusev synthesized via co-precipitation method. The A549 (human lung carcinoma) cells were purchased in Russian collection of The Loginov Moscow Clinical Scientiﬁc Center, Moscow, Russia. vertebrate cell cultures, Russian Academy of Sciences, St. 2 3 Kazan Federal University, Kazan, Russia. Graduate School of Petersburg, Russia. We utilized transmission electron microscopy Medicine, Juntendo University, Tokyo, Japan. Endocrinology method in order to visualize the cell uptake process. PVP and PEI Research Centre, Moscow, Russia; ORCID: 0000-0003-0673-3590 polymers were used for conjugation procedure. The UV laser was used as a substitution of X-ray irradiation. Introduction: Approximately 5 to 10% of cancer cases are Results: The efﬁciency of the conjugation was controlled by associated with hereditary cancer predisposition syndromes shortening the lifetime curves of the doping ions. The efﬁcient (HCPS). Early identiﬁcation of HCPS is important for the choice radii between doping ions and radaclorin molecules were in the of the individual monitoring and treatment strategies. 5.0 – 7.1 nm range. The conjugates are non-toxic in micromolar Materials and methods: Study cohort included 1119 probands concentrations toward A549 cells. Both PVP – and PIE-based from Russia: 1062 (94.9%) cancer patients with clinical signs of conjugated are effectively uptaken by A549 cells via micropino- HCPS and 57 (5.1%) healthy individuals with cancer in family cytosis. The conjugated are packed into 200 – 1500 nm vesicles. history. NGS analysis of 76 HCPS genes was performed using a According to the ﬂow cytometry measurements, the intensity of custom Roche NimbleGen panel, which encompassed exonic and side scattered light (SSC) of PIE-based conjugates in two times intronic regions of the genes. bigger compared to PVP-based one. It reveals, that the PIE-based Results: We found that 34.0% (381/1119) of the patients conjugates are uptaken more effectively. enrolled had pathogenic/likely pathogenic variants. 54 (27.4%) out Conclusions: Taking into consideration the fact the PVP-based of total 197 unique genetic alterations hadn’t been previously conjugates are non-stable and they form precipitate on the 3+ 3+ described in the literature, 9 (4.6%) out of these variants were bottom of the beaker, the Ce ,Tb :YF nanoparticle-PVP located in BRCA1/BRCA2 genes. The predominant number (59.3%) -“radaclorin” conjugates are more effective for X-ray induced of genetic variants was identiﬁed in the BRCA1/BRCA2 genes photodynamic therapy. associated with breast and ovarian cancer syndromes. CHEK2 was Funding: This work was carried out in accordance with the the second most commonly altered gene with the total of 29 Strategic Academic Leadership Program "Priority 2030" of the (7.6%) identiﬁed variants: 4 likely pathogenic and 25 pathogenic Kazan Federal University of the Government of the Russian variants. 155 (40.6%) genetic variants were found in other 35 Federation. cancer-associated genes of variable penetrance: 12 (3.1%) variants Disclosure: The authors declare no competing interests. Cell Death Discovery (2022) 8:1 – 11 7 Efﬁcacy of drugs targeting mitochondria in inhibiting the Bioenergetic heterogeneity of commonly used glioma cell proliferation of colorectal cancer cells lines 1 1 2 1 1 2 Gulnaz Sharapova , Sirina Kurbangaleeva , Nikita Markov , Sirina Kurbangaleeva , Gulnaz Sharapova , Nikita Markov , 1 3 1 3 1 Marina Gomzikova , Anna Brichkina , Albert Rizvanov, Hans- Marina Gomzikova , Anna Brichkina , Albert Rizvanov , Hans- 1,2 1,2 Uwe Simon Uwe Simon Laboratory of Molecular Immunology, Kazan Federal University, Laboratory of Molecular Immunology, Kazan Federal University, Kazan, Russia; Institute of Pharmacology, University of Bern, Bern, Kazan, Russia; Institute of Pharmacology, University of Bern, Bern, Switzerland; Center for Tumor and Immune Biology, Philipps Switzerland; Center for Tumor and Immune Biology, Philipps University of Marburg, Marburg, Germany; ORCID: 0000-0002- University of Marburg, Marburg, Germany; ORCID: 0000-0002- 9404-7736 9404-7736 Introduction: Gliomas are brain tumors originating from glial Introduction: Mitochondrial aberrations are often found in cells and are characterized by poor prognosis and very limited tumor cells of different origin, including colorectal cancer treatment options. Developing of new therapeutic options to (CRC). Mitochondrial dysfunctions are usually associated treat gliomas depends on the understanding of their molecular with the inability of the electron transport chain (ETC) to properties and features.Currently, numerous drugs targeting sustainhighlevelsofoxidative phosphorylationdue to the different cellular metabolic and bioenergetic axes of cancer impaired activity of ETC complexes. Moreover, impaired cells are undergoing clinical trials. However, the efﬁciency of ETC often leads to severe adjustments within the TCA cycle these drugs is highly dependent on the bioenergetic proﬁles leading to the accumulation of fumarate. In this work, we and dependencies of examined cancer cells. In this study, explored how pharmacological induction of mitochondrial we investigated the metabolic proﬁlesofcommonlyused dysfunction and the subsequent switch to glycolysis, induced glioma cell lines to select for potential therapeutic by treatment of cells with the ETC complex I and III inhibitors targets. Moreover, our study proposes a new level of actinomycin A and rotenone, respectively, affects the characterization of glioma cells separating them by their survival and proliferation of CRC cells. In addition, we bioenergetic capacity. evaluated the effect of increased concentrations of fumarate by treating CRC cells with the cell permeable derivate dimethyl Materials and methods. In order to obtain bioenergetic fumarate. proﬁles of six glioblastoma (A172, LN428, D247MG, LN18, LN229, Materials and methods. Crystal violet staining of viable cells T98G) and two astrocytoma (U251, LN319) cell lines, we utilized was implemented and their proliferation rates were measured the Seahorse XFe96 analyzer. The assay was performed upon using CellTrace Violet assay. Additional experiments were subsequent injections of oligomycin,FCCP,rotenone/actinomy- performedinstarvationconditions (2% FBS) or during the cin A and 2-DG. This design of assay allowed us to calculate 9 growth of the cells in spheroids. Drugs were tested in the parameters related to glycolysis and mitochondrial respiration. concentration range of 156 nM – 2 μM on four different CRC cell To minimize the interference of external factors, the metabolic lines: SW480, SW620, SW48 and HCT-15 during a time period of proﬁles of all 8 cell lines were measured simultaneously. The 0-96 hours. CRC cell lines were chosen based on different number of seeded cells varied in the range of 40 to 60 thousand bioenergetic axes and characterized by different levels of owing to their difference in size. The ﬁnal results were mitochondrial activity. normalized using the initial seeding coefﬁcients. In order to Results: Drugs targeting mitochondrial complexes of the avoid additional variability induced by an uneven proliferation ETC limited proliferation of CRC cell lines within both rate of examined cell lines, the cells were seeded on poly-L- nanomolar and micromolar ranges of concentrations. More- lysine coated plates allowing for rapid adhesion and immediate over, the effect of the decreased proliferation of cancer cells execution of the assay. was observed in both glycolytic cells (HCT-15) and cells relying Results: Bioenergetic proﬁling of examined cells separated on mitochondrial respiration (SW480), suggesting that cancer them into three distinct clusters. Within the ﬁrst cluster, the cell cells are sensitive to the ETC targeting independently of their lines A172, LN319, LN428, T98G were characterized by both high metabolic programs. On the other hand, treatment with levels of glycolysis and mitochondrial respiration. In contrast, dimethyl fumarate did not affect the proliferation of CRC cell lines D247MG, LN18, LN229 were less metabolically active cell lines (SW620, SW48, HCT-15). Interestingly, dimethyl and displayed attenuated levels of glycolysis and mitochondrial fumarate at nanomolar concentrations promoted a minor activity. Interestingly, the highest level of glycolysis and increase in proliferation of SW480 cells, suggesting a possible relatively low mitochondrial respiration was detected in U251 modulating effect of fumarate on the cell cycle. In addition, cell line suggesting the presence of mitochondrial dysfunction the observed effect was conﬁrmed in the settings of in these cells. Further studies are required to understand spheroid growth and starvation (2% FBS), suggesting that how bioenergetic clustering ﬁts current molecular and WHO- dimethyl fumarate indeed upregulates proliferation of based classiﬁcations of glioma and correlates with such SW480 cells. parameters as survival, progression of the disease and sensitivity Conclusion: Targeting of mitochondrial ETC complexes appears to treatments. to be an effective strategy to limit the expansion and growth of Conclusion: Simultaneous measurement of bioenergetic pro- CRC cells independently of their metabolic proﬁles. In contrast, ﬁles is a powerful tool allowing to investigate the heterogeneity of dimethyl fumarate either does not affect the proliferation rates of cancer cells and cluster them by their metabolic activity. CRC cells or promotes a minor increase in proliferation of some Commonly used glioma cell lines are characterized by distinct cancer cell lines like SW480. bioenergetic programs. Funding: This work was supported by the grant from Russian Funding: This work was supported by the grant from Russian Government #075-15-2021-600. Government #075-15-2021-600. Disclosure: The authors declare no competing interests. Disclosure: The authors declare no competing interests. Cell Death Discovery (2022) 8:1 – 11 8 Inhibition of TGF-β receptors of bladder cancer cells affects UCNP-based Photoluminescent Nanomedicines for their mechanical properties as measured by Theranostics of HER2-positive tumors nanopipette–based scanning ion-conductance microscopy 1 1 1 Evgenii L. Guryev , Daria K Bausheva , Natalia Y Shilyagina , 1 2,3 1,2 2 3 Irina V. Balalaeva I , Sergey M. Deyev S.M , Andrei V Anastasiia Sapach , Roman A. Akasov , Nikita Savin , Petr 1,3,4 3 1,4,5 2 Zvyagin Gorelkin , Gleb B Sukhorukov , Andrei V Zvyagin Lobachevsky State University of Nizhny Novgorod, Nizhny Nov- Skolkovo Institute of Science and Technology, Moscow, Russia; 2 3 gorod, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Sechenov First State Medical University, Moscow, Russia; The Chemistry, Russian Academy of Sciences, Moscow, Russia; Institute National University of Science and Technology MISIS, Moscow, 4 5 of Molecular Theranostics, Sechenov University, Moscow, Russia; MQ Russia; Siberian State Medical University, Tomsk, Russia; Queen Photonics Centre, Macquarie University, Sydney, Australia; ORCID: Mary University of London, London E1 4NS, United Kingdom; ORCID: 0000-0001-8799-2257 0000-0002-5745-2177 Introduction: Development of new approaches for diagnosis and Introduction: Epithelial–mesenchymal transition (EMT) is a therapy of tumors (taken together, termed theranostics) - one of fundamental process that governs dissemination of single the most dynamic areas of the life sciences, where new carcinoma cells from the primary tumors. Remodeling of the nanomaterials afford new opportunities. In virtue of their unique actin cytoskeleton as well as modulation of extracellular matrix optical properties, upconversion nanoparticles (UCNP) have occurs within EMT and is associated with cell stiffness. Cell shown promise for cell and live animal imaging. A combination stiffness is usually measured by atomic force microscopy that of agents, a bacterial exotoxin PE40 genetically fused with a generally needs cell ﬁxation and labor-intensive sample prepara- targeting protein DARPin (targeted toxin DARPin-LoPE) and tion. The aim of this research was to study mechanical properties radioactive beta-emitters Y were assembled into an UCNP of live bladder cancer cells when blocking EMT by inhibition of nanocomplex that was characterized and deployed in cancer TGF-β receptors by using nanopipette–based scanning ion- treatment experiments to exhibit a profound super-additive conductance microscopy (SICM). therapeutic effect in cells and live animal models. The binding Materials and methods: T24 cell line established from a speciﬁcity of as-produced theranostic nanomedicine and its toxic human urinary bladder cancer patient was employed in the effect on HER2-positive tumor cells were studied and reported. experiment. 5 × 10 cells were treated with 10 µM of inhibitors (A-83-01 or A-77-01) and 5 ng/ml of TGF-β in 24 h, and, following Materials and methods: UCNPs of the structure NaYF :Yb:Tm washes, were scanned at room temperature. Cell topology and were coated with a two-layer shell of organic polymers. The stiffnessmapswereacquiredusing SICM (ICAPPIC ltd, UK) by targeted toxin DARPin-LoPE was attached to polymer-coated sampling cells as reported elsewhere. A nanopipette were used UCNPs by chemical conjugation. The developed conjugation as the probe pulled from borosilicate blanks, outer/inner protocol enabled achievement of the desired orientation of the diameters, 1.2 mm/0.69 mm, respectively, to produce nanopip- shell molecules and protein modules and preserve their functional ettes with the radius ranged from 40 to 50 nm. Topography and activity. The speciﬁcity of the interaction of UCNP complexes with measurements of the mechanical properties were recorded in SKOVip-kat human ovarian adenocarcinoma cells was studied “hopping mode” over areas of 40 × 40 µm with the resolution using ﬂuorescence microscopy, while the cytotoxicity was 0.3 µm. Setpoint for topography was 0.5% and for the stiffness assessed by an MTT method. mapping 1 % and 2 %. Image processing was performed using Results: The mean hydrodynamic diameter and zeta potential the «SICMImageViewer» software. of as-produced UCNP-DARPin-LoPE complexes were measured to Results: Stiffness measurement of single cell mechanical be 234 ± 29 nm and -54 ± 9 mV, respectively, indicating colloidal properties via Scanning Ion-conductance Microscopy is a novel stability. The UCNP-DARPin-LoPE complexes were designed to method, which enables simultaneous topography mapping target HER2-positive tumor cells due to speciﬁc interaction with and stiffness mapping. Cell stiffness of TGF-β-induced/inhibi- target molecules on their surface. The role of these molecules is tion-blocked EMT of bladder cancer cells was measured using the HER2 tumor marker receptor. DARPin protein exhibited high SICM. Control cell stiffness measurement showed 1.7±0.2 kPa, afﬁnity to the epidermal growth factor receptor and served as the when TGF-β treated cells decreased value (0.3±0.05 kPa). targeting agent to HER2-expressing cells. To demonstrate the Cells incubated with 10 µM of inhibitors (A-83-01 or A-77-01) selective accumulation of UCNP-DARPin-LoPE-s on the HER2- and 5 ng/ml of TGF-β were signiﬁcantly (p<0.05) stiffer (0.7±0.1 expressing cell surface, we employed ﬂuorescence microscopy kPa) than cells incubated with 5 ng/ml of TGF-β only. The tuned to excite and acquire UCNP photoluminescence signal, cell height exhibited similar trend, i.e., cells incubated with 5 where SKOVip-kat cells transfected to express far-red ﬂuorescence ng/ml TGF-β featured higher proﬁle than these of negative proteins were used as the HER2-positive tumor cell model. No control or TGF-β/A-83-01 treated cells. Cells incubated with UCNP cell binding was detected in negative controls, including 10 µM of inhibitors (A-83-01 or A-77-01) maintained their polymer-coated UCNPs with no targeting protein decoration and membrane stiffness level even in the presence of 5 ng/ml HER2-negative cells. The cytotoxicity study showed a pronounced of TGF-β. combined effect of the Y beta-emitters and the DARPin-LoPE Conclusions: SCIM was successfully employed as a method for toxins on SKOVip-kat cells overexpressing the HER2 receptor. studying mechanical properties of living T24 cells with activation/ Preliminary in vivo experiments showed selective accumulation of inhibition of EMT. UCNP-DARPin-LoPE-s in peritoneal tumor foci in mice formed by Funding: The work was funded by the Siberian State SKOVip-kat cells. Medical University’s development program Priority 2030 Conclusions: Theranostic UCNP-DARPin-LoPE nanocomplexes and by the Ministry of Science and Higher Education of were able to selectively bind to tumor cells overexpressing the the Russian Federation, the contract 075-15-2021-709, RF- HER2 receptors owing to a DARPin targeting module. Two toxic 2296.61321X0037. modules, Y beta-emitters and the DARPin-LoPE-s conspired Disclosure: The authors declare no competing interests. to achieve a combined therapeutic effect. The obtained results Cell Death Discovery (2022) 8:1 – 11 9 showed potential of UCNP-DARPin-LoPE nanocomplexes diagno- Introduction: The role of p53 in autoimmune diseases has been sis and therapy of HER2-positive tumors. demonstrated in many models. Multiple sclerosis (MS) is a chronic Funding: This work was supported by Ministry of Education and autoimmune disease that affects myelin. Currently, there are 44 Science of the Russian Federation: UNN “Priority 2030” Strategic new cases of MS per 100,000 population worldwide diagnosed Academic Leadership Program (Project No. H-418-99_2022-2023). every year, and the number of patients is constantly growing. Disclosure: The authors declare no competing interests. However, the role of p53 in the regulation of MS is still unclear. In this study we evaluated the effect of p53 activator Nutlin-3a on cytokine secretion by immune cells obtained from a healthy Reactivation of tumor suppressor p53 by aminobenzothiazole donor and an MS patient. derivatives Materials and methods. We used peripheral blood mono- nuclear cells (PBMCs) of a healthy donor and an MS patient and 1 1 1 divided them into 3 groups: untreated, treated with 10 μM and Raniya Khadiullina , Damir Davletshin , Elvina Khusainova , 1 1 2 40 μM Nutlin-3a. PBMCs were incubated with Nutlin-3a for Regina Mirgayazova , Vitaly Chasov , Matthias Baud , Emil 24 hours. To evaluate the effect of Nutlin-3a on p53 activation Bulatov and expression of p53-dependent genes we assessed p21, Bax and 1 PUMA expression levels by quantitative RT-PCR. In addition, ﬂow Kazan Federal University, Institute of Fundamental Medicine and 2 cytometry analysis was carried out for cells stained with antibodies Biology, Kazan, Russia. University of Southampton, Department of to CD45, CD3, CD4, CD20 and p53 protein. Cell supernatants were Chemistry, Southampton, United Kingdom; ORCID: 0000-0003-3521- collected and tested by cytokine multiplex analysis using Bio-Plex 200 system (Bio-Rad, USA). Results and conclusion. RT-PCR analysis demonstrated Introduction: The TP53 gene is mutated in all human cancers and increased expression levels p53-dependent genes (p21, Bax, the p53 protein is an essential component of the cell response PUMA). Flow cytometry analysis revealed increased proportion of induced by various genotoxic stresses. The presence of mutations p53 positive T- and B-cells both in cells from healthy donor and violates the tertiary structure of the DNA-binding domain p53, which MS patient after co-incubation of PBMCs with Nutlin-3a. At the leads to destabilization of the protein, its partial denaturation and same time, the ratio of the analyzed populations of immune cells loss of activity. A promising therapeutic strategy for treating a wide did not change signiﬁcantly. According to multiplex cytokine range of human malignancies involves using small molecule analysis, in healthy donor cells Nutlin-3a increased the level of therapeutics to reactivate mutant p53. First, we evaluated the IP-10, reduced IL-10, G-CFS, MCP-1, TNF-α, VEGF and did not affect cytotoxic effect of aminobenzothiazole derivatives on normal IL-1b, IL-1ra, IL-15, INF-γ, RANTES. In MS patient cells, Nutlin-3a human ﬁbroblast cell line (HSF) and four human adenocarcinoma increased the levels of IL-1b and TNF-α, decreased IL-1ra, IL-10, cell lines with various p53 status (wild type: A549; mutant: MIA G-CSF, and VEGF and did not affect IL-15, IFN-γ, IP-10, MCP-1, p53(R248W) p53(R248Q) p53(−/−) PaCa-2 ,OVCAR-3 ; and knockout: MCF-7 ). RANTES. Our results show that activation of p53 by Nutlin-3a Afterthatwe identiﬁed gene expression alterations in human affects cytokine secretion in PBMCs of both healthy donor and MS p53 signaling pathway induced by these compounds. patient. This suggests a potential role of p53 protein in regulation Materials and methods: Cells were treated with compounds at of immune-related processes in MS. a concentration range of 2.5-120 μM or 1% DMSO (vehicle control) Funding: This work was supported ﬁnancially by the Strategic and assessed after 24 h and 48 h using colorimetric MTS assay. Academic Leadership Program of Kazan Federal University Quantitative RT-RCR and western blotting were performed (PRIORITET-2030). according to standard protocol after treatment cell lines with Disclaimer: The authors declare no conﬂict of interest. 60 μM compounds or vehicle control for 48 h. Results. MTS assay revealed a signiﬁcant dose- and time-dependent cytotoxic effect of the compounds on all cell lines. However, this effect was more Pharmacological activity of invertebrate steroid hormone, pronounced in cancer cells than in normal cells, and cancer cells ecdysterone, in human normal and cancer cells with p53 wild-type or knockout tended to be less sensitive than those with mutant p53. Furthermore, the compounds showed upregulated p53 target gene transcription in cancer cells with Oleg Shuvalov, Yuilia Kirdeeva, Elyzaveta Feﬁlova, Alexandra mutant p53. At the same time no substantial modulating effects Daks, Olga Fedorova were observed in cell line with wild-type p53. Conclusions: Our study helped to further reveal molecular Institute of Cytology RAS, St Petersburg, Russia; ORCID: mechanisms underlying the reactivation of different mutant p53 forms. This will be important for the development of novel, Introduction: In human, the insect hormone 20-hydroxyecdysone individually tailored anticancer drugs. (ecdysterone) displays a number of beneﬁcial pharmacological Funding: The study was funded by RSF grant 22-24-20034 and activities including antioxidant, hypoglycemic, hepato-, cardio- strategically supported by Kazan Federal University Strategic and neuroprotective. Ecdysterone is broadly known among Academic Leadership Program (PRIORITY-2030). athletes for its anabolic activity, due to which it is used worldwide Disclosure: The authors declare no competing interests. to increase physical ﬁtness. In addition, a number of studies has recently shown its antineoplastic activity. However, to date, there is no understanding of either the molecular mechanisms or mode Effect of p53 activator Nutlin-3a on populations of immune of ecdysterone’s activity in different types of cells including cells obtained from healthy donor and patient with multiple malignant ones. sclerosis Materials and methods: We have carried out the comparison of ecdysterone-induced biological effects in mouse (C2C12) and Irina Ganeeva, Aygul Valiullina, Ekaterina Zmievskaya, Albert rat (L8) myoblasts, mouse embryonic ﬁbroblasts (MEFs), human Rizvanov, Emil Bulatov ﬁbroblasts (DF2) and non-small cell lung adenocarcinoma cell lines (H460, H1299, A549 and H1975) in a dose-dependent Institute of Fundamental Medicine and Biology, Kazan Federal manner. We have studied the inﬂuence of ecdysterone on their University, Kazan, Russia; ORCID: 0000-0003-4041-5000 proliferation, cell cycle, level of reactive oxygen species (ROS), Cell Death Discovery (2022) 8:1 – 11 10 membrane potential of mitochondria, energy metabolism, autop- Introduction: Receptor tyrosine-protein kinase ERBB3, also known hagy and the resistance to chemotherapeutic drugs. as human epidermal growth factor receptor 3 (HER3), is a gene Results: Ecdysterone has slightly decreased the proliferation of that encodes a member of the epidermal growth factor receptor all cells. It signiﬁcantly reduced glycolysis and respiration and (EGFR) family. Preclinical studies demonstrated that mutations in induced autophagy. At the molecular level, ecdysterone strongly ERBB3 gene can promote oncogenesis in a ligand-independent suppressed the expression of c-Myc and some glycolytic enzymes manner in breast cancer (ВС). Five missense mutations in ERBB3 which are its transcriptional targets. In addition, ecdysterone are associated with breast neoplasms. Furthermore, HER3 over- notably augmented ROS production in cancer models whereas expression is associated with poor prognosis in patients both with reduced it in normal cells. HER2-positive and the triple-negative breast cancer (TNBC). Conclusions: Ecdysterone suppresses energy metabolism in Germline genetic variants in ERBB3 affect response to therapy cancer cells and induce ROS production. Due to safety and agents in HER2-positive BC patients. The aim of the study was to plethora of valuable pharmacological properties, ecdysterone determine the germline genetic variants of ERBB3 gene in TNBC should be in focus of researches as potential adjuvant which may patients, study the correlation with overall and progression-free help cancer patients to prevail consequences of chemotherapy as survival. well as make it more efﬁcient. Materials and methods: The study involved 20 patients with Funding: This work was supported by the RSF grant No. 21-75- TNBC and the control group, consisting of 21 volunteers without 10138. family history of cancer in three generations. Genomic DNA was Disclosure: The authors declare no competing interests. isolated from peripheral blood mononuclear cells. Exome sequen- cing was performed on Illumina’s NextSeq platform using SureSelect clinical research exome V2 system (Agilent, USA). Regulation of autophagy ﬂux by E3 ubiquitin ligase Pirh2 Statistical data processing was carried out using a chi-square test and Kaplan-Mayer survival analysis. Samples were collected 1 1 1 according to KFU Ethical Committee-approved protocol. Alexandra Daks , Sergey Parfenyev , Oleg Shuvalov , 1 1 1 Results: We have identiﬁed three SNPs rs773123, rs56017157, Anastasia Gudovich , Nick Barlev , Olga Fedorova rs2271188 in patients with TNBC and healthy volunteers. The rate 1 of rs773123 genetic variant was signiﬁcantly higher in TNBC Institute of Cytology, Russian Academy of Sciences, 194064, St compared to control group (p = 0.007). Presence of rs773123 did Petersburg, Russian Federation; ORCID: 0000-0003-1382-4204 not affect overall survival (p = 0,644) in our study. Missense- mutation rs773123 in ERBB3 gene leads to S1119C protein change. Introduction: Autophagy is a catabolic process aimed at the Several studies conﬁrm that presence of SNP rs773123 was restoration of the energy supply in cells under various forms of associated with worse relapse-free survival, decreased sensitivity stress. Initially, autophagy was considered as one of the cell death to doxorubicin, carboplatin and trastuzumab in HER2-positive BC mechanisms and only later it was recognized as a protective patients. mechanism. Human Pirh2 (p53-induced RING-H2 protein is an E3 Conclusions: Germline single nucleotide polymorphism ubiquitin ligase that mediates p53 degradation. In addition to p53, rs773123 in ERBB3 might be associated with increased risk of Pirh2 was shown to attenuate Chk2, p73, p63, polH, c-myc, and triple-negative breast cancer. p27 thus Funding: This work was part of Kazan Federal University regulating cell proliferation, cell cycle progression, and DNA Strategic Academic Leadership Program (PRIORITY-2030). repair. Disclosure: The authors declare no competing interests. Materials and methods: We cultivated tumor cells with knockdown of Pirh2 and control. The level of autophagy was evaluated by western blot analysis followed by qPCR analysis of Genetic varians of DNA repair genes in cisplatin-resistant marker genes. ovarian cancer cells Results: We have revealed E3 ligase, Pirh2, that affects the expression of different genes that participate in all steps of 1 1 2 autophagy. Using Western blot and immunoﬂuorescent analysis Aisylu Sagdeeva , Julia Filina , Yana Shamsutdinova , Rezeda we demonstrated that Pirh2 positively regulates the accumulation 2 2 1 Galimova , Alexey Sabirov , Aigul Rakhmatullina , Maria of LC3-II in cancer cell lines. 1 1 1 Zolotykh , Rimma Mingaleeva , Albert Rizvanov , Regina Conclusions: We have uncovered a novel function of Pirh2 in Miftakhova the regulation of autophagy in cancer cells. Funding: This work was supported by the grant from RSF No. Institute of Fundamental Medicine and Biology, Kazan Federal 18-75-10076. University, Kazan, Russia; Tatarstan Regional Clinical Cancer Center, Disclosure: The authors declare no competing interests. Kazan, Russia; ORCID: 0000-0002-9318-2004 Germline genetic variants of ERBB3 in triple-negative breast Introduction: High activity of the DNA repair system deﬁned by cancer the genetic variants may contribute to the chemotherapy resistance, which is one of the most common complications in 1 2 1,3 the ovarian cancer treatment. The aim of the study was to Maria Zolotykh , Alfya Nesterova , Airat Bilyalov , Albert 2 1 1 1 dedermine the clinically signiﬁcant single nucleotide polymorph- Gimranov , Julia Filina , Eugenia Boulygina , Albert Rizvanov , isms involved in ovarian cancer development and chemotherapy Regina Miftakhova resistance. Materials and methods: FFPE surgical resections and blood Institute of Fundamental Medicine and Biology, Kazan Federal samples (n = 86) of OC patients were obtained from the Tatarstan University, Kazan, Russian Federation, Tatarstan Regional Clinical Regional Clinical Cancer Center. All patients underwent cisplatin Cancer Center, Kazan, Russian Federation, Moscow Clinical Research monotherapy (n = 42) after surgery and were observed for at least Center named after A.S. Loginov MHD, Moscow, Russian Federation; 6 months after the end of chemotherapy. According to the ORCID: 0000-0003-2473-5514 disease-free interval patients were divided into platinum-resistant Cell Death Discovery (2022) 8:1 – 11 11 (disease progression during therapy or within 6 months after) and Conclusions: Polymorphic variants GG444 of the APEX1 gene platinum-sensitive (patients without relapse for more than and GG977 of the OGG1 gene may be promising biomarkers for 6 months after therapy). Control blood samples (n = 37) were predicting the effectiveness of therapy and ovarian cancer obtained from healthy volunteers without cancer cases in outcome. three generations. DNA was isolated from sections of the normal Funding: This work was supported by the Russian Foundation tissue of patients with ovarian cancer and control blood samples for Basic Research grant 17-00-00263 and performed in accor- to assess germinal mutations. Genomic DNA sequencing was dance with the Kazan Federal University Strategic Academic carried out using the target NGS panel covering 20 genes of the Leadership Program (PRIORITY-2030). DNA repair and replication system coding regions. Statistical Disclosure: The authors declare no competing interests. analysis was performed using Fisher’s exact test; p≤0.05 was considered statistically signiﬁcant. Samples were collected accord- Open Access This article is licensed under a Creative Commons ing to KFU Ethical Committee-approved protocol. Attribution 4.0 International License, which permits use, sharing, Results: Sequencing analysis revealed 356 single nucleotide adaptation, distribution and reproduction in any medium or format, as long as you give variants in 20 genes of the DNA repair and replication system in appropriate credit to the original author(s) and the source, provide a link to the Creative healthy volunteers and patients with OC. Eleven SNVs in ATM, Commons license, and indicate if changes were made. The images or other third party BRCA1, FEN1, MUTYH, POLI and POLQ were associated with the material in this article are included in the article’s Creative Commons license, unless ovarian cancer development. We also observed higher rate of indicated otherwise in a credit line to the material. If material is not included in the germline APEX1 c.T444G SNV in platinum-resistant compared to article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly platinum-sensitive OC patients: homozygous GG genotype was from the copyright holder. To view a copy of this license, visit http:// more frequent in the resistant patients in the comparison with TT creativecommons.org/licenses/by/4.0/. +TG (p = 0.045). OGG1 c.C977G genotype was associated with disease-free survival in OC: median time was 0.756 months for GG and 11.441 months for CC and CG carriers (p = 0.009). © The Author(s) 2022 Cell Death Discovery (2022) 8:1 – 11
Cell Death Discovery – Springer Journals
Published: Dec 14, 2022
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