Research group-Malgorzata Czystowska-Kuzmicz, PhD

                                                                                                                

Our research focus on better understanding of the influence of small extracellular vesicles (sEVs)/exosomes on immune cells, in particular in the context of the development of cancer. We investigate how the cancer cells utilizise sEVs to supress the immune response and how it may impact potential immunotherapy. Performing quantitative and qualitative analyses of sEVs from biological specimens of cancer patients, we try to determine their diagnostic or prognostic potential as so called ”liquid biopsy”. Furthermore we investigate the immunosupressive mechanisms induced by sEVs in in vitro co-cultures of murine/human immune cells with EVs as well as in murine tumor models. We extensively collaborate with clinical groups: Chair and Department of Internal Diseases, Pulmunology and Allergology of Medical University of Warsaw, Depatment of Gynecology and Gynecologic Oncology of Military Institute of Medicine, Warsaw, Institute of Tuberculosis and Lung Diseases, Warsaw, Department of Obsterics and Gynecology, Praski Hospital, Warsaw.  

 

Research

  • Optimalisation of isolation methods of sEVs/exosomes from patients’ biological material (plasma, ascites, bronchoalveolar lavage fluid) for their identification as potential cancer biomarker: size exclusion chromatography (SEC), immunomagnetic isolation
  • Implementation and optimalization of modern analysis teqhniques of sEVs/exosomes: fluorescence Nanoparticle Tracking Analysis (FL-NTA), flow cytometry using beads coated with antibodies against exosome marker
  • The impact of sEVs on the metabolism of amino acids in the tumor microenviroment of ovarian cancer
  • Role of sEVs as carrier of icheckpoint molecules in the supression of anti-tumor
  • Correlation between the individual molecular profile of sEVs and the extend of immune suppression in patients with non small-cell lung cancer
  • The impact of sEVs on immunotherapy, in particular therapies using immune checkpoint inhibitors

Biography

Małgorzata Czystowska-Kuzmicz graduated from the Department of Biology of the Heinrich-Heine University in Düsseldorf (Germany) in 2001. She performed her PhD studies in the Laboratory of Molecular Genetics, Department of Obstetrics and Gynecology, Heinrich-Heine University, Duesseldorf and received her doctoral degree in biological sciences in 2006. In the years 2006-2010 she continued her work as a postdoctoral fellow at one of the leading laboratories in EV research, at the University of Pittsburgh Cancer Institute (USA) of prof. Theresa Whiteside. After maternal leave in 2013 she joined the Department of Immunology, Medical University of Warsaw, where she worked within the multidisciplinary BASTION (From Basic to Translational Research in Oncology) project (2013-2015). Then she established her own research team at the Immunology Department, where she implements research projects funded by the Polish National Science Center and the National Agency of Academic Exchange. In 2019 she moved to the Chair and Department of Biochemistry at the Medical University of Warsaw, where she continues her research.

 

Research team

PhD students: 

Magdalena Dlugolecka, MSc

Since 2019, she has been a participant of the Doctoral School of the Medical University of Warsaw and a scholarship holder in the team of PhD Małgorzata Czystowska-Kuźmicz in the project “Molecular profile of exosomes found in the fluid from bronchoalveolar lavage as a new biomarker of immune response impairment in non-small cell lung cancer”, financed by the National Science Centre in the Opus competition. She isolates and characterizes small extracellular vesicles (EVs) from patients with non-small cell lung cancer. EVs are isolated from the plasma of patients by Size ExclusionChromatography (SEC) and from bronchoalveolar lavage fluid (BALF) by ultracentrifugation. The main techniques he uses to characterize vesicles are fluorescent Nanoparticle tracking Analysis (FL-NTA), flow cytometry using magnetic beads, western blot, and Elisa.

Karolina Soroczynska, MSc

Students:

Lukasz Zareba, MD

Since 2020, he has been a scholarship holder in the team of PhD Małgorzata Czystowska-Kuźmicz in the project “Molecular profile of exosomes found in the fluid from bronchoalveolar lavage as a new biomarker of immune response impairment in non-small cell lung cancer”, financed by the National Science Center in the Opus competition. He isolates and characterizes small extracellular vesicles from patients with non-small cell lung cancer using methods such as SEC, isolation via ultracentrifugation, and NTA. Since 2021, he has been a participant of the Doctoral School of the Medical University of Warsaw. In his doctoral project, he experiments on mice models and uses techniques such as immunohistochemistry and western blot. 

Technicians: Iwona Rosa

Former team members

Senior scientists: Marta Szajnik, PhD

Post-docs: Magdalena Grazul, PhD

Technicians: Zuzanna Homoncik, MSc

Students: Jacek Szymański

 

Ongoing projects

Contribution of extracellular vesicles and exosomal arginase to immune dysfunction in endometriosis

Opus, National Science Center, duration: 2022-2026

Objective:

One of the most prevailing theories of endometriosis origin is the so called retrograde menstruation theory, when the menstrual blood reaches through the fallopian tubes into the pelvic cavity, allowing the implantation of the in this way co-transported endometrial cells at sites distant from the uterus (ectopic endometrium). The phenomenon of retrograde menstruation occurs however in most females of reproductive age and it is not known why only some women develop endometriosis. Accumulating research studies are indicating a connection between the development of endometriosis and a dysfunction of the immune system, which is eliminating ectopic endometrial cells at normal conditions. Changes in numbers and dysfunction of different immune cell populations, like cytotoxic T-cells, NK cells and macrophages, have been observed in women with endometriosis. A well know immunosuppressive factor, especially within the tumor microenvironment, is arginase (ARG), one of the enzymes of the amino acids metabolism. We have recently discovered elevated levels of the two arginase isoforms, ARG1 and ARG2, as well as increased arginase activity in serum of endometriosis patients compared to healthy women. Furthermore, in previous studies we provided the first evidence for the presence of ARG1 in tumor-derived EVs – nano-sized double-membrane-enclosed particles released by tumor cells, in ovarian cancer. Hereby we identified a new mechanism of tumor-induced immunosuppression, based on the delivery of ARG1 by EVs to local lymph nodes, where the enzyme blocks T-cell activation and proliferation. A similar, however only temporary, down-regulation of T-cell activity as in tumors has been observed in the female reproductive tract (FRT) during the menstrual cycle and normal pregnancy. This T-cell inhibition correlated with an increased expression of ARG in the endometrial tissue. Furthermore, evidence is accumulating that analogous to the key role of EVs in pre-metastatic niche formation and metastatic organotropism in cancers, also EVs in the uterine and peritoneal microenvironment may support implementation and growth of ectopic endometrial cells outside the uterus.

Therefore, we hypothesize that a similar immunosuppressive mechanism mediated by ARG+ EVs, as described by us in OvCa, may play a role in the promotion of endometriosis and may be responsible for the observed dysfunction of several types of immune cells in this disease. The aim of the project is to identify arginase (ARG)-carrying extracellular vesicles (EVs) in the serum and peritoneal fluid (PF) of endometriosis patients and to decipher their impact on the dysfunction of the immune system in endometriosis and to verify their diagnostic or prognostic potential. Basing on our previous profound expertise in studying EVs, we will use state-of -the-art methods for phenotyping and functional analysis of EVs isolated from serum and PF. We will try to answer the question if the clinically relevant differences in the EV profile and the ARG-EV content found in PF are also reflected in the EV profile from peripheral blood. If yes, this EV profile could be translated into a clinically useful diagnostic test in the future. We will determine the effects of EV-associated ARG on selected types of immune cells like cytotoxic T-cells, NK cells and macrophages in functional cell co-cultures. Since exosomal arginases, as we have shown in OvCa, are more stable, easily cross tissue barriers and are transported over long distances, they may exert systemic biological effects and significantly contribute to niche formation for ectopic lesions and disease progression. Therefore, we believe that they can be a more significant and reliable potential biomarker than soluble arginases. Besides, ARG is a promising therapeutical target and has already entered clinical trials in cancer treatment. One can assume, that similar therapeutic options may become available for endometriosis, once the role of arginases in the progression of the disease will be confirmed. We believe, that this project will be a first step in this direction.

Molecular profile of exosomes in the bronchoalveolar lavage fluid as a new biomarker for impaired immune response in non-small cell lung cancer

OPUS, National Science Center, duration: 2018-2022

Objective:

Our own previous studies as well as studies from other researchers point out the key role of small EVs in the modulation of anti-tumor response. Small EVs, as one of the key elements of intercellular communication within the tumor microenvironment and carrier of genetic material and functional suppressive molecules derived from the tumor, are an essential mediator of local and systemic immune suppression. Currently many studies explore exosomes from peripheral blood as easy accessible sources of biomarkers of the patient’s immune status, which allow to choose the optimal treatment and to monitor treatment response. However the changes detected in peripheral blood may not reflect the actual changes within the tumor microenvironment, which are significant for treatment. The aim of our project is to elucidate if the exosome profile in systemic circulation reflects the profile of exosomes from the tumor microenvironment and may be a marker of immune dysfunctions within the tumor lesion in non small-cell lung cancer (NSCLC).

An innovative aspect of the project is the implementation of bronchoalveolar lavage (BAL) as a method for the isolation of exosomes from the tumor microenvironment. It is a low-invasive, standardized procedure which can be performed during diagnostic bronchofiberoscopy routinely done for very patient with suspected lung cancer .We plan to perform a comprehensive analysis of the molecular profile of exosomes present in BAL fluid in reference to the immune status within the tumor microenvironment of NSCLC patient.

We assume that the individual molecular profile of exosomes present in the tumor microenvironment will significantly affect the local immune response as well as the response to potential immunotherapy, which we want to check in a murine model of lung cancer. We hope that the in-depth molecular and functional analysis of BALF exosomes will contribute towards the identification of exosomes as tumor markers and provide a target for novel anti-cancer strategies.

Finished projects:

Elucidation of the role of tumor-derived and exosomal arginases in avoiding immune responses by ovarian cancer

OPUS, National Science Center, duration:2014-2018

In this project we provided the first evidence for the role of ARG1-containing EVs in the formation of an immunosuppressive microenvironment in ovarian cancer. ARG1 is an enzyme involved in amino acid metabolism We showed that small EVs found in the ascites and plasma of OvCa patients contain ARG1.These EVs distribute ARG1 from tumor cells to antigen-presenting cells in secondary lymphoid organs, suppressing antigen-specific T-cell proliferation and activation. High ARG1 expression in primary tumors and increased ARG1 activity in plasma correlated with worse prognosis in OvCa patients. In an OvCa mouse model, we showed that blocking arginase activity mitigates ARG1-driven tumor progression. We identified hereby a novel mechanism of tumor-induced systemic T-cell dysfunction based on the activity of tumor-derived ARG1+ EVs, that may also apply to other arginase-expressing tumor types and may have significant clinical implications for T-cell immunotherapy approaches.

Publications:

Dlugolecka M, Szymanski J, Zareba L, Homoncik Z, Domagala-Kulawik J, Polubiec-Kownacka M, Czystowska-Kuzmicz M. Characterization of Extracellular Vesicles from Bronchoalveolar Lavage Fluid and Plasma of Patients with Lung Lesions Using Fluorescence Nanoparticle Tracking Analysis. Cells. 2021 Dec 9;10(12):3473. doi: 10.3390/cells10123473. PMID: 34943982; PMCID: PMC8699990.

Zareba L, Szymanski J, Homoncik Z, Czystowska-Kuzmicz M. EVs from BALF-Mediators of Inflammation and Potential Biomarkers in Lung Diseases. Int J Mol Sci. 2021 Apr 1;22(7):3651. doi: 10.3390/ijms22073651. PMID: 33915715; PMCID: PMC8036254.

Czystowska-Kuzmicz M, Whiteside TL. The potential role of tumor-derived exosomes in diagnosis, prognosis, and response to therapy in cancer. Expert Opin Biol Ther. 2021 Feb;21(2):241-258. doi: 10.1080/14712598.2020.1813276. Epub 2020 Sep 8. PMID: 32813990; PMCID: PMC7880862.

Czystowska-Kuzmicz M, Sosnowska A, Nowis D, Ramji K, Szajnik M, Chlebowska-Tuz J, Wolinska E, Gaj P, Grazul M, Pilch Z, Zerrouqi A, Graczyk-Jarzynka A, Soroczynska K, Cierniak S, Koktysz R, Elishaev E, Gruca S, Stefanowicz A, Blaszczyk R, Borek B, Gzik A, Whiteside T, Golab J. Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma. Nat Commun. 2019 Jul 5;10(1):3000

Sosnowska A, Czystowska-Kuzmicz M, Golab J. Extracellular vesicles released by ovarian carcinoma contain arginase 1 that mitigates antitumor immune response. Oncoimmunology. 2019 Aug 20;8(11):e1655370.

Szajnik M, Czystowska-Kuźmicz M, Elishaev E, Whiteside TL. Biological markers of prognosis, response to therapy and outcome in ovarian carcinoma. Expert RevMol Diagn. 2016 Aug;16(8):811-26.

Czystowska M, Gooding W, Szczepanski MJ, Lopez-Abaitero A, Ferris RL, Johnson JT, Whiteside TL. The immune signature of CD8(+)CCR7(+) T cells in the peripheral circulation associates with disease recurrence in patients with HNSCC. Clin Cancer Res. 2013 Feb 15;19(4):889-99

Rahma OE, Ashtar E, Czystowska M, Szajnik ME, Wieckowski E, Bernstein S, Herrin VE, Shams MA, Steinberg SM, Merino M, Gooding W, Visus C, Deleo AB, WolfJK, Bell JG, Berzofsky JA, Whiteside TL, Khleif SN. A gynecologic oncology group phase II trial of two p53 peptide vaccine approaches: subcutaneous injection and intravenous pulsed dendritic cells in high recurrence risk ovarian cancer patients. Cancer Immunol Immunother. 2012 Mar;61(3):373-84.

Czystowska M, Szczepanski MJ, Szajnik M, Quadrini K, Brandwein H, Hadden JW, Whiteside TL. Mechanisms of T-cell protection from death by IRX-2: a new immunotherapeutic. Cancer Immunol Immunother. 2011 Apr;60(4):495-506

Szajnik M, Czystowska M, Szczepanski MJ, Mandapathil M, Whiteside TL. Tumor-derived microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg). PLoS One. 2010 Jul 22;5(7):e11469.

Czystowska M, Strauss L, Bergmann C, Szajnik M, Rabinowich H, Whiteside TL. Reciprocal granzyme/perforin-mediated death of human regulatory and responder T cells is regulated by interleukin-2 (IL-2). J Mol Med (Berl). 2010

Mandapathil M, Hilldorfer B, Szczepanski MJ, Czystowska M, Szajnik M, Ren J, Lang S, Jackson EK, Gorelik E, Whiteside TL. Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells. J Biol Chem. 2010 Mar 5;285(10):7176-86.