Raman imaging - a new method for characterizing lymphoma cells

Patrycja Dawiec¹, Patrycja Leszczenko¹, Anna M. Nowakowska¹, Paulina Laskowska², Aleksandra Borek-Dorosz¹, Adriana Adamczyk¹, Justyna Jakubowska⁴, Marta Ząbczyńska⁴, Agata Pastorczak⁴, Kinga Ostrowska⁴, Maciej Szydłowski², Piotr Mrówka^2,5, Małgorzata Barańska^1,3, Katarzyna Majzner^1,3

¹Faculty of Chemistry, Jagiellonian University, Poland

²Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Poland

³Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Poland

⁴Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Poland

⁵Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Poland

Form: poster presentation

How to recognize polarized from unpolarized macrophages? Raman analysis

Magdalena Cudak¹,  Aleksandra Borek-Dorosz¹,  Anna M. Nowakowska¹, Paulina Laskowska²,  Ewa Kurtz², Maciej Szydłowski^2, Piotr Mrówka^2,3, Małgorzata Barańska^1,4, Katarzyna Majzner^1,4

¹Jagiellonian University, Faculty of Chemistry, Krakow, Poland

²Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Poland

³Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Poland

⁴Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Poland

Form: poster presentation

Raman imaging proves differences in lipid metabolism of ruxolitinib-treated acute lymphoblastic leukemia cells

Maja Bartoszek¹, Adriana Adamczyk¹, Anna M. Nowakowska¹, Justyna Jakubowska², Marta Ząbczyńska², Agata Pastorczak², Kinga Ostrowska², Małgorzata Barańska^1,3, Katarzyna Majzner^1,3

¹Jagiellonian University, Faculty of Chemistry,  Gronostajowa 2, 30-387, Krakow, Poland

²Medical University of Lodz, Department of Pediatrics, Oncology and Hematology, Sporna 36/50, 91-738 Lodz, Poland

³Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzyńskiego 14, 30-348 Krakow, Poland

Form: poster presentation

Raman spectroscopy-based classification of selected Acute Lymphoblastic Leukemia molecular subtypes 

A. Adamczyk¹, A. Dorosz¹, P. Leszczenko¹, A. Fatla¹,  S. Kashyrskaya¹, A. Grabacka¹, M. Bartoszek¹, A. M. Nowakowska¹, J. Jakubowska³, A. Pastorczak³, K. Ostrowska³, M. Ząbczyńska³, K. Majzner^1,2, M. Barańska^1,2

¹Jagiellonian University, Faculty of Chemistry, Gronostajowa 2 str. 30-387 Krakow, Poland

²Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14 str. 30-348 Krakow, Poland

³Medical University of Lodz, Department of Pediatrics, Oncology and Hematology, Sporna 36/50 str , 91-738 Lodz, Poland

Form: poster and flash presentation

Is Raman imaging a reliable tool for genetic subtypes discrimination  of B-type acute lymphoblastic leukemia?

P. Leszczenko¹, A. Borek-Dorosz^1,2, A. M. Nowakowska¹, A. Adamczyk¹, S. Kashyrskaya¹, J. Jakubowska³, M. Ząbczyńska³, A.  Pastorczak³, K. Ostrowska³, M. Barańska^1,2, K. M. Marzec⁴, K. Majzner^1,2

¹Jagiellonian University, Faculty of Chemistry, Raman Imaging Group, Gronostajowa 2, 30-387 Krakow, Poland;

²Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, 30 348 Krakow, Poland;

³Medical University of Lodz, Department of Pediatrics, Oncology and Hematology, Sporna 36/50, 91-738 Lodz, Poland;

⁴Lukasiewicz Research Network – Krakow Institute of Technology, Zakopiańska 73, 30-418 Krakow, Poland

Form: poster presentation

Protocol for Raman analysis of leukemia cells

A. M. Nowakowska¹, A. Borek-Dorosz^1,2, P. Leszczenko¹, A. Adamczyk¹, A. Pieczara², J. Jakubowska³, K. Ostrowska³, A. Pastorczak³, K. Brzozowski1, M. Barańska^1,2, K. Maria Marzec², K.Majzner^1,2*

¹Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Street, 30-387 Krakow, Poland

²Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Street, 30-348 Krakow, Poland

³Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Sporna Street 36/50 Łodz, Poland

Form: poster presentation

Metabolic changes identified in IDH1 and IDH2- mutated transgenic  leukemic cells using Raman imaging

A. M. Nowakowska¹,  P. Laskowska², A. Borek-Dorosz^1,3, J. Stolarska¹, A. Adamczyk¹, P. Leszczenko¹, M. Zasowska², M.Szydłowski², E. Białopiotrowicz², P. Mrówka^2,4, M. Barańska^1,3, K. Majzner^1,3

¹Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Street, 30-387 Krakow, Poland

²Department of Experimental  Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland

³Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Street, 30-348 Krakow, Poland

⁴Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Warsaw, Poland

Form: poster and flash presentation

Identification of Raman spectroscopic signature of IDH mutations in AML in vitro model

P. Laskowska¹, A. Nowakowska², S. Orzechowska², A. Borek-Dorosz^{2, 3}, J. Stolarska², A. Adamczyk², P. Leszczenko², M. Zasowska¹, A. Szlachetka¹, M. Szydłowski¹, E. Białopiotrowicz¹, P. Juszczyński¹, M. Barańska^{2, 3}, K. Majzner^{2, 3,} P. Mrówka^{1, 4}

¹Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Warsaw,

²Faculty of Chemistry, Jagiellonian University, Krakow

³Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow,

⁴Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Warsaw, Poland

Labelled-Spontaneous and Stimulated Raman Spectroscopy to image selected molecules in living cancer cells

Adriana Adamczyk¹, Anna M. Nowakowska¹,  Krzysztof Brzozowski¹, Justyna Jakubowska²,  Katarzyna Majzner^1,3, Malgorzata Baranska^1,3

¹Jagiellonian University, Faculty of Chemistry, Gronostajowa 2 str. 30-387 Krakow, Poland
²Medical University of Lodz, Department of Pediatrics, Oncology and Hematology, Sporna 36/50 str , 91-738 Lodz, Poland
³Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14 str. 30-348 Krakow, Poland

Form: oral presentation

Raman imaging as a tool to identify lymphoma cells

Patrycja Dawiec¹, Patrycja Leszczenko¹, Anna M. Nowakowska¹, Paulina Laskowska², Aleksandra Borek-Dorosz^1,3, Adriana Adamczyk¹, Justyna Jakubowska⁴, Marta Ząbczyńska⁴, Agata Pastorczak⁴, Kinga Ostrowska⁴, Maciej Szydłowski², Piotr Mrówka^2,5,
Małgorzata Barańska^1,3, Katarzyna Majzner^1,3

¹Faculty of Chemistry, Jagiellonian University,  Poland
²Department of Experimental  Hematology, Institute of Hematology and Transfusion Medicine, Poland
³Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Poland
⁴Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Poland;
⁵Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Poland

Form: poster presentation

Distinguishing different phenotypes of polarized macrophages by Raman spectroscopy

Magdalena Cudak¹,  Aleksandra Borek-Dorosz¹,  Anna M. Nowakowska¹, Paulina Laskowska², Maciej Szydłowski², Piotr Mrówka^2,3,  Małgorzata Barańska^1,4, Katarzyna Majzner^1,4

¹Jagiellonian University, Faculty of Chemistry, Krakow, Poland
²Department of Experimental  Hematology, Institute of Hematology and Transfusion Medicine, Poland
³Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Poland
⁴Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Poland

Form: poster presentation

Spectroscopic characteristics of the interaction of the JAK2 kinase inhibitor ruxolitinib with the in vitro model
of acute lymphoblastic leukemia

Maja Bartoszek¹, Adriana Adamczyk¹, Anna M. Nowakowska¹,  Justyna Jakubowska², Marta Ząbczyńska², Agata Pastorczak², Kinga Ostrowska², M. Barańska^1,3, K. Majzner^1,3

¹Jagiellonian University, Faculty of Chemistry, Krakow, Poland
²Medical University of Lodz, Department of Pediatrics, Oncology and Hematology, Lodz, Poland
³Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland

Form: poster presentation

Charakterystyka chłoniaka rozlanego z dużych komórek B metodą obrazowania ramanowskiego

P. Dawiec¹, P. Leszczenko¹, A. M. Nowakowska¹, P. Laskowska², A. Borek-Dorosz^1,3, A. Adamczyk¹, J. Jakubowska⁴, M. Ząbczyńska⁴, A. Pastorczak⁴, K. Ostrowska⁴, M. Szydłowski², P. Mrówka^2,5, M. Barańska^1,3, K. Majzner^1,3

¹Uniwersytet Jagielloński, Wydział Chemii, Kraków, Polska
²Instytut Hematologii i Transfuzjologii, Warszawa, Polska
³Uniwersytet Jagielloński, Jagiellońskie Centrum Rozwoju Leków, Kraków, Polska
⁴Uniwersytet Medyczny w Łodzi, Klinika Pediatrii, Onkologii, Hematologii, Łódź, Polska
⁵Warszawski Uniwersytet Medyczny, Zakład Biofizyki, Fizjologii i Patofizjologii, Warszawa, Polska

Form: oral presentation

Diffuse large B-cell lymphoma (DLBCL) subtypes distinguished using Raman imaging

P. Dawiec¹, P. Leszczenko¹, A. M. Nowakowska¹, P. Laskowska², A. Borek-Dorosz^1,3, A. Adamczyk¹, J. Jakubowska⁴, M. Ząbczyńska⁴, A. Pastorczak⁴, K. Ostrowska⁴, M. Szydłowski², P. Mrówka^2,5, M. Barańska^1,3, K. Majzner^1,3

¹Faculty of Chemistry, Jagiellonian University, Poland
²Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Poland
³Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Poland
⁴Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Poland;
⁵Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Poland

Form: poster presentation

Accumulation of lipids and carotenoids as a marker of leukocytes activation studied by spontaneous and stimulated Raman Spectroscopy

A. Borek-Dorosz^1,2, A. M. Nowakowska¹, P. Laskowska³, K. Brzozowski¹, W. Tipping⁵, M. Cudak¹, M. Zasowska³,  M. Szydłowski³, P. Mrówka^3,4, D. Graham⁵, P. Juszczyński³, M. Baranska^1,2, K. Majzner^1,2*

¹Jagiellonian University, Faculty of Chemistry, Krakow, Poland

²Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland

³Department of Experimental  Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland

⁴Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Warsaw, Poland

⁵Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, United Kingdom

Activation of leukocytes is an important step in inflammation, which in turn causes
a cascade of molecular and biochemical changes inside the cells. Inflammatory conditions can lead to different molecular response regarding the type of leukocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells develop from hematopoietic stem cells in lymphopoiesis process [1] and to become fully functional effector cells their activation is required. T cell activation requires extracellular stimulatory signals that are mainly mediated by T cell receptor (TCR) complexes that is a complex of integral membrane proteins participating in response to an antigen. T cells activation involves a series of biochemical intracellular events leading to production subtype specific effector proteins and accelerated proliferation [2].

Macrophages as different type of leukocytes which develop from myeloid progenitor and differentiate from monocytes. Macrophages display a high plasticity, which allows them to adapt their phenotype in response to different environmental stimuli. Two major polarization states have been described for macrophages, the classically activated type 1 (M1) and the alternatively activated type 2 (M2), that differ in the types of secreted cytokines and biological functions and is related with the process by which macrophages produce distinct functional phenotypes as a reaction to specific microenvironmental stimuli and signals [3]. Understanding of activation processes and related with polarization biochemical changes in macrophages will help for better understanding how activated macrophages regulate the physiology and pathology state.

In our studies we applied a label-free spontaneous Raman imaging and Stimulated Raman Scattering (SRS) methods for molecular characterization and discrimination of activated states in leukocytes (Fig. 1). We have defined spectral biomarkers which can be used to differentiate between normal and activated cells. The accumulation of carotenoids found exclusively in T cells, but not B cells, was clearly evidenced in the Raman spectra and supported by SRS. For reliable discrimination between studied types of macrophages polarization
a detailed analysis of the average spectra with application of PCA and PLS methods was applied.

References

[1] 5. Hematopoietic Stem Cells | stemcells.nih.gov". stemcells.nih.gov. Retrieved 2020-11-21.

[2] D. G. Waller, A. P. Sampson (2018) Rheumatoid arthritis, other inflammatory arthritis’s and osteoarthritis, Medical Pharmacology and Therapeutics (Fifth Edition), Elsevier

[3] Y. Yongle et al. (2019) Macrophage Polarization in Physiological and Pathological Pregnancy, Front. Immunol. 10:792. Doi: 10.3389/fimmu.2019.00792

Keywords: activated T cells, macrophage polarization, spontaneous Raman spectroscopy, Stimulated Raman Scattering, carotenoids, lipids

Acknowledgments

This work was supported by „Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net program of the Foundation for Polish Science co-financed by the EU under the ERDF.

Form: oral presentation

Labelled-Spontaneous Raman and Stimulated Raman Scattering on the track of chemical and morphotic changes related to induced differentiation of promyeloblastic in vitro model

A. Adamczyk¹, A. M. Nowakowska¹, K. Brzozowski¹, J. Jakubowska³,  K. Majzner^1,2, M. Barańska^1,2

¹Jagiellonian University, Faculty of Chemistry, Gronostajowa 2 str. 30-387 Krakow, Poland

²Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14 str. 30-348 Krakow, Poland

³Medical University of Lodz, Department of Pediatrics, Oncology and Hematology, Sporna 36/50 str, 91-738 Lodz, Poland

Despite of many advantages related to the label-free spectroscopic determination of markers of biochemical and morphological changes in single cells, the concept of molecular Raman reporters is gaining importance. Well-designed Raman probes, containing triple bonds in their structure or isotopically substituted compounds, together with a selective targeting group with an affinity towards cellular compartments, display low bandwidth signals allowing multiple cell organelle imaging. The relevance of this approach could be additionally enhanced by the development of non-linear Raman-based techniques that enable a significant increase of signal intensity with simultaneously reduced integration time and enhanced sensitivity of imaging.

Promyeloblasts originated from hematopoietic stem cells, are produced in the bone marrow and under physicochemical conditions they transform into functional and mature blood cells like neutrophils, eosinophils, and monocytes. This process is characterized by changes in the assembly of essential biochemical compounds or partial/complete loss of the cell compartment functions. Thus, the first signs of differentiation can be detected by studying the organelles’ state and the ability of cells to sustain their proper functions. Herein, we evaluated an in vitro model of induced differentiation by all-trans retinoic acids (ATRA) towards neutrophil-like cells using human leukemia cell line HL-60. Changes associated with mitochondria and nucleus were followed using MitoBady and 5-Ethynyl-2'-deoxyuridine (EdU) by detecting Raman signal of molecular Raman reporters at 2120 and 2220 cm-1, respectively. 

Keywords: Cell silent region, Raman reporters, myeloid induced differentiation, leukemia

Acknowledgments

This work was supported by „Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net program of the Foundation for Polish Science co-financed by the EU under the ERDF.

Form: oral presentation

Raman imaging-based analysis of acute lymphoblastic leukemia  with KMT2A gene rearrangement

M. Bartoszek¹, A. Adamczyk¹, A. M. Nowakowska¹, J. Jakubowska³, M. Ząbczyńska³, A. Pastorczak ³, K. Ostrowska³, M. Barańska^1,2, K. Majzner^1,2*

¹Jagiellonian University, Faculty of Chemistry, Krakow, Poland

²Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland

³Medical University of Lodz, Department of Pediatric, Oncology and Hematology, Lodz, Poland

The predominant type of pediatric cancers in group 0-19 years are leukemias with frequency rate ~30%. The most common type (75-85%) is definitely acute lymphoblastic leukemia (ALL) with average peak of occurrence between 3-5 years old. ALL cells might harbor harmful structural genomic alterations, which can generate relapse of the primary disease. Among these genomic lesions prominent place belongs to histone-lysine N-methyltransferase 2A (KMT2A) formerly known as MLL. Chromosomal translocations involving KMT2A gene, are the most common abnormalities in infants and represent extremely aggressive subtype of leukemia, hallmarked with poor prognosis. Moreover, the KMT2A chromosomal translocations are cytogenetically heterogenous and still hard to treat with available therapies.

Currently, apart from seeking for new treatment approaches of blood malignancies, significant progress in development of novel diagnostic methods supporting rapid and accurate diagnosis is visible.  One of such analytical techniques, providing information about the molecular structure of cells is Raman spectroscopy. Each biological sample has a unique spectroscopic profile that enables determining specific differences in their biochemical compositions. Moreover, in combination with chemometric tools, it enables precise discrimination between healthy cells and their malignant counterparts. 

The main aim of this study was to distinguish precursor B-cell ALL (BCP-ALL) with KMT2A chromosomal translocations from healthy lymphocytes B, using Raman spectroscopy. Two BCP-ALL cell lines carrying KMT2A chromosomal rearrangements (SEM-K2, RS4;11) were used, and measurement were carried out using 532 nm and 633 nm laser excitation wavelengths. Principal component analysis (PCA) was performed on obtained Raman spectra to reduce the number of variables describing the trends in the analyzed dataset. The study showed significant differences at the biochemical level between normal lymphocytes B from healthy donors and leukemic cells. The spectra of lymphocytes B, were characterized by the presence of intense bands that originated mainly from bonds vibrations in DNA and from nitrogenous bases (1580, 785 cm-1). Raman spectra of healthy lymphocytes B were distinct from both examined leukemic cell lines, for which significant intense bands characteristic for unsaturated lipids (at 3015, 1660, and 1267 cm-1) were observed.

Keywords: Raman Imaging, Acute Lymphoblastic Leukemia; KMT2A gene rearrangement.

Acknowledgments

The „Label-free and rapid optical imaging, detection and sorting of leukemia cells” project is carried out within the Team-Net program of the Foundation for Polish Science co-financed by the EU under the ERDF.

Form: abstract publication

Spectroscopic discrimination between B- and T-cell acute lymphoblastic leukemia

K. Czuja¹, P. Leszczenko¹, A. M. Nowakowska¹, A. Borek-Dorosz^1,2, J. Jakubowska³, M. Ząbczyńska³, A. Pastorczak³, K. Ostrowska³, M. Barańska^1,2, K. Majzner^1,2*

¹Faculty of Chemistry, Jagiellonian University, Krakow, Poland

²Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland

³Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Lodz, Poland;

Acute lymphoblastic leukemia (ALL) is a clonal lymphoid malignancy derived from B- and T-lymphoid progenitors.  ALL can be broken into subtypes depending on whether genetic alteration occurred in a developing T-lymphocytes or B-lymphocytes and further classified into distinct molecular subtypes depending on specific gene rearrangements. Since different subtypes of ALL are characterized by unique gene expression profile and chemosensitivity, identification and classification of prognostic relevant genetic changes is important in order to assess patients risk stratification and intensity of treatment. Standard diagnostic scheme of ALL is based on morphologic, immunophenotypic and genetic features, allowing for appropriate distinction between normal progenitors and different hematopoietic malignancies.

In our study, we used Raman spectroscopy for biochemical and molecular characterization of leukemic cells, within clinical material from patients suffering from different subtypes of ALL. Raman spectra of leukemic cells were compared with spectra of normal B and T lymphocytes from healthy donors, which constituted a control group. Using Raman imaging, chemometric methods (k- means cluster analysis (KMCA) and principal components analysis (PCA)) B-ALL cells were successfully distinguished from T-ALL ones based on differences in their spectral profiles. Moreover, leukemic cells were successfully differentiated from their healthy counterparts.

Summarizing, our study revealed diagnostic potential of Raman spectroscopy as sensitive and precise tool for identification of B- and T-ALL cells.

Keywords: Raman imaging; acute lymphoblastic leukemia (ALL); B cell; T cell; chemometrics;

Acknowledgments

The „Label-free and rapid optical imaging, detection and sorting of leukemia cells” project is carried out within the Team-Net program of the Foundation for Polish Science co-financed by the EU under the ERDF.

Form: abstract publication

Application of Raman spectroscopy to characterize B-cell lymphoma

P. Dawiec¹, P. Leszczenko¹, A. M. Nowakowska¹, P. Laskowska², A. Borek-Dorosz^1,3, A. Adamczyk¹, J. Jakubowska⁴, M. Ząbczyńska⁴, A. Pastorczak⁴, K. Ostrowska⁴, M. Szydłowski², P. Mrówka^2,5, M. Barańska^1,3, K. Majzner^1,3

¹Faculty of Chemistry, Jagiellonian University, Poland

²Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Poland

³Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Poland

⁴Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Poland

⁵Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Poland

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma in adults. It accounts for approximately 30% - 40% of non-Hodgkin's lymphomas and over 80% of the aggressive lymphomas. DLBCL is a genetically very heterogeneous group, but some subsets share molecular features that can be revealed by gene expression or metabolic profiles. While B-cell receptor (BCR)-dependent DLBCLs exhibit high glycolytic flux (non-OxPhos), OxPhos-DLBCLs rely on mitochondrial energy and are independent from BCR signaling and are characterized by increased oxidative phosphorylation. Currently, these two subtypes have been distinguished based on morphological, cytogenetic, immunohistochemical and genetic characteristics. However, new tools are sought to improve the stage of lymphoma diagnosis to improve treatment outcome. The goal of the research was to identify spectroscopic profile which can be used to discriminate DLBCL cells and normal B lymphocytes, as well as the overall Raman-based characterization of biochemical composition of selected subsets of DLBCLs.

The in vitro model of lymphoma was constituted from four DLBCL cell lines: OCI-Ly1, OCI-Ly7, Pfeiffer, and Toledo. In addition, normal B lymphocytes, isolated from the peripheral blood of healthy donors, were used as control group. Raman imaging followed by chemometric methods have been applied in the research in order to reveal unique spectroscopic profile of DLBCL cells. A WITec Alpha 300 confocal Raman system with two excitation wavelengths (532 nm and 633 nm) was used for spectroscopic imaging. Data pre-processing and k-means cluster analysis (KMCA) were done using Project Five 5.1 Plus software. Another chemometric method, principal component analysis (PCA), was performed using the Unscrambler X software.

PCA performed on averaged spectra of normal B cells and DLBCL cells clearly showed that the two groups are different .. The spectra of tumor cells mainly consisted of bands characteristic of lipids and proteins, while B cells were characterized mostly by bands derived from nucleic acids.

Summing up, Raman spectroscopy is a technique that can differentiate DLBCL tumor cells from normal lymphocytes. This method also allowed for the biochemical characterization of DLBCL cells. The metabolic differences of the studied DLBCL subtypes and normal B cells were reflected on Raman spectra, due to which their characteristic spectroscopic markers were found.

Keywords: Raman spectroscopy, lymphomas, DLBCLs, B cells, chemometrics

Acknowledgments

The „Label-free and rapid optical imaging, detection and sorting of leukemia cells” project is carried out within the Team-Net program of the Foundation for Polish Science co-financed by the EU under the ERDF.

Form: abstract publication

Spectroscopic characteristics of Philadelphia-positive acute lymphoblastic leukemias

A. Fatla¹, A. Adamczyk¹, A. M. Nowakowska¹, J. Jakubowska³, M. Ząbczyńska³, A. Pastorczak³, K. Ostrowska³, K. Majzner^1,2*

¹Jagiellonian University, Faculty of Chemistry, Gronostajowa 2, 30-387 Krakow, Poland

²Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland

³Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Poland

Acute lymphoblastic leukemia (ALL) is a clonal, hematological malignancy originating from aberrant lymphoid progenitor cells accumulated within the bone marrow. Cytogenetic analyses revealed that the most characteristic hallmarks of ALL are structural and copy number genomic alterations within bone marrow lymphoid precursors, which in consequence cause maturation disorder and uncontrollable proliferation. An average 85% of ALL cases is represent by B-cell precursor ALL (BCP-ALL) classified into diverse molecular subtypes. BCP-ALL cells might harbor structural genomic alterations, which are assigned as a prone to be more chemoresistant and therefore generate relapse of the primary disease. Among these genomic lesions important position belongs to BCR–ABL1 [Philadelphia chromosome (Ph) t(9;22)(q34;q11)-positive] being one of the most recurrent disease-initiating genetic alteration in BCP-ALL. Ph-positive ALL, being significantly associated with poor prognosis owe this unfavorable phenomenon to constitutive activity of the BCR-ABL1 oncoprotein. This aberrant tyrosine kinase transfers a phosphate group to target molecules and modifies
a phosphorylation pattern in multiple signaling pathways, strongly enhancing proliferation of leukemic cells. Therefore, identification of the Philadelphia chromosome is important for accurate determination of risk stratification and intensity of treatment.

The research hypothesis of this work was based on the presence of spectroscopic markers enabling identification of the Philadelphia chromosome. For this purpose, the following cell lines were tested: K562 (CML + BCR-Abl, Ph+), TOM-1 (ALL + BCR-Abl, Ph+), SD-1 (pre-B-ALL + BCR-Abl, Ph+). In order to identify spectral fingerprints of Ph+ due to Raman imaging approach, two excitation laser lines (532nm and 633nm) was used. Data interpretation was done based on the analysis of characteristic Raman bands, clustering methods (KMCA, HCA) and principal component analysis (PCA).

Summarizing, obtained results allowed for simultaneous spectroscopic characterization of Ph+ cell lines and detection of differences related with type of leukemia (chronic myeloid leukemia (CML) vs ALL). Our results indicated promising potential of Raman imaging for the identification of clinically relevant subtypes of leukemia carrying BCR-Abl fusion gene.

Keywords: Raman spectroscopy, chemometrics, leukemia, Philadelphia chromosome, BCR-Abl fusion gene

Acknowledgement

This work was supported by ‘‘Label-free and rapid optical imaging, detection and sorting of leukemia cells” project, which is carried out within the Team-Net programme (POIR.04.04.00-00-16ED/18-00) of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Form: abstract publication

Raman microscopy as a supportive tool in diagnostics of acute lymphoblastic leukemia subtypes

P. Leszczenko¹, A. Borek-Dorosz^1,2, A. M. Nowakowska¹, A. Adamczyk¹, S. Kashyrskaya¹, J. Jakubowska³, M. Ząbczyńska³, A. Pastorczak³, K. Ostrowska³, M. Barańska^1,2, K. M. Marzec4, K. Majzner^1,2*

¹Jagiellonian University, Faculty of Chemistry, Gronostajowa 2, 30-387 Krakow, Poland

²Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland

³Medical University of Lodz, Department of Pediatrics, Oncology and Hematology, Sporna 36/50, 91-738 Lodz, Poland

⁴Lukasiewicz Research Network – Krakow Institute of Technology, Zakopiańska 73, 30-418 Krakow, Poland

Despite significant progress within treatment of blood malignancies, leukemia still remains the 11th leading cause of cancer-related mortality worldwide. Leukemia refers to clonal blood malignancy hallmarked by transformed hematopoietic progenitor cells and diffuse infiltration of bone marrow. The most common type of childhood lymphoid neoplasm is undeniably acute lymphoblastic leukemia (ALL) derived from B - and T-lymphoid progenitors. B-cell precursor ALL (BCP-ALL) accounts for 85% of total ALL cases and is classified in to several subtypes, characterized by unique gene expression profile and diverse clinical and biological outcomes. Raman imaging merged with chemometric analysis has a great potential in order to support the routine diagnostics of leukemia. Raman spectroscopy as a non-destructive tool allows for measurement of cells in their natural environment in a label-free manner.

Herein, we used Raman spectroscopy to scrutinize the biochemical composition of healthy B lymphocytes (control group) and their malignant counterparts belonging to B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) [1]. We have analyzed three selected molecular subtypes of BCP-ALL defined by gene rearrangements: BCR-ABL1, TCF3-PBX1, and TEL-AML1. Using numerous chemometric tools such as Principal Component Analysis (PCA) we were able to discriminate studied molecular subtypes of BCP-ALL cells from healthy B cells based on the differences in their biochemical profile. Moreover, by means of Partial Least Squares Discriminant Analysis (PLS-DA) we managed to train an algorithm to not only differentiate healthy B cells from malignant ones but also determine whether the cells carry the BCR-ABL1 or TCF3-PBX1 gene rearrangement. Unfortunately, the above-mentioned approach, was not sufficient in identification of cells with TEL-AML1 fusion gene, which require application of more advanced supervised teaching methods in order to improve developed algorithm. We have found that molecular fingerprint of TEL-AML1 cells is heterogenous, therefore biochemical differences reflected in Raman spectra between this BCP-ALL leukemia subtype and two other studied (BCR-ABL1 and TCF3-PBX1) were very subtle. Nevertheless, obtained results clearly indicate that Raman spectroscopy supported by chemometric analysis has the potential to become a powerful tool supporting the diagnosis of leukemia.

References

[1] Leszczenko, P. et al., Towards Raman-based screening of acute lymphoid leukemia-type B (B-ALL) subtypes, Cancers, 2021, 13(21), 5483.

Keywords: Raman spectroscopy, chemometrics, acute lymphoblastic leukemia, BCR-ABL1, TCF3-PBX1, TEL-AML1

Acknowledgement

This work was supported by ‘‘Label-free and rapid optical imaging, detection and sorting of leukemia cells” project, which is carried out within the Team-Net programme (POIR.04.04.00-00-16ED/18-00) of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Form: abstract publication

How important is the Raman imaging sample preparation protocol in the diagnosis of leukemia?

A. M. Nowakowska¹,  A. Borek-Dorosz^1,2, P. Leszczenko¹, A. Adamczyk¹, A. Pieczara², J. Jakubowska³, K. Ostrowska³, A.Pastorczak³, K. Brzozowski¹, K. Maria Marzec⁴, K.Majzner^1,2,*

¹Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Street, 30-387 Krakow, Poland

²Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Street, 30-348 Krakow, Poland

³Department of Pediatric, Oncology, Hematology and Diabetology, Medical University of Lodz, Sporna Street 36/50 Lodz, Poland

⁴Lukasiewicz Research Network-Krakow Institute of Technology, Zakopiańska 73, 30-418 Krakow, Poland

Cancer remains second most common cause of death in children with over 1,500 deaths annually. By far the most common type of childhood cancer is leukemia representing nearly 33% of all malignancies in children. Despite progress in cancer research accurate diagnosis and treatment are still one of the major challenges of health care. In order to support and improve standard diagnostic pipeline of leukemia, novel techniques and methods are needed. Raman spectroscopy may be considered as efficient technique supporting diagnosis of leukemia, due to high chemical sensitivity and label-free detection of single living cells in a non-destructive manner. Moreover, great advantage of this method consist of the possibility to detect signature of biochemical and metabolic changes during malignant transformation with resolution of single cells. However, high chemical sensitivity of Raman imaging also directs attention to the high importance of sample preparation, which can significantly affect appropriate identification of leukemic cells [1–3]. Therefore, the aim of our studies was simultaneous optimization of sample preparation methodology and verification of its impact on confocal Raman imaging of leukemic cells including their biochemical composition, in the context of clinical practice [4].

Our studies were focused on testing both the influence of glutaraldehyde (GA) fixation [5] (0.1%, 0.5% and 2.5% GA), and sample storage on biochemical profile of T cell acute lymphoblastic leukemia (T-ALL) and healthy peripheral blood mononuclear cells (PBMCs) with Raman imaging. T-ALL and PBMCs were imaged using WITec Alpha300 system equipped with two lasers, 532 nm and 633 nm. Obtained Raman images were subjected to k – means cluster analysis (KMCA), and further average spectra were analyzed using principal component analysis (PCA). Results of our studies revealed changes in the protein secondary structure due to GA fixation, detected by the increase of the intensity of the band at 1041 cm-1, characteristic for phenylalanine [5,6]. Moreover, we found that GA at a concentration 0.5% was the most optimal for fixation purpose both in the case of healthy and cancer cells. We also verified the chemical stability of fixed cells within 11 days of storage. Summarizing, our results indicated that chemical procedures applied in the preparation of cells may affect Raman spectra and therefore significantly influence the identification of spectroscopic markers characteristic for different subtypes of leukemia.

References

[1] A.J. Hobro et al., Vib. Spectrosc. 91 (2017) 31–45.

[2] A.D. Meade et al., Anal. Bioanal. Chem. 396 (2010) 1781–1791.

[3] E. Gazi et al., Biopolymers. 77 (2005) 18–30.

[4] N. Chaudhary et al., Anal. Methods. (2021)

[5] E. Bik et al., Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 240 (2020) 118460.

[6] B. Hernández et al. J. Raman Spectrosc. 44 (2013) 827–833.

Keywords: glutaraldehyde fixation, Raman imaging, T-ALL, PBMCs

Acknowledgments

The „Label-free and rapid optical imaging, detection and sorting of leukemia cells” project is carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the EU.

Form: abstract publication

Transfected HEL cells with IDH2 mutations –  a confocal Raman spectroscopy study

J. Stolarska¹, A. M. Nowakowska¹, A. Borek-Dorosz^1,2, P. Laskowska³, A. Adamczyk¹, P. Leszczenko¹, M. Zasowska³, Maciej Szydłowski³, E. Białopiotrowicz³, P. Mrówka^3,4, K. Majzner^1,2*

¹Faculty of Chemistry, Jagiellonian University, Krakow, Poland

²Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland

³Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland

⁴Department of Biophysics, Physiology and Pathophysiology, Medical University of Warsaw, Warsaw, Poland

Acute myeloid leukemia (AML) is one of the most common types of myeloid malignancies diagnosed in adults and the risk of getting it increases with the age of the patient [1]. Mutations in genes IDH1 and IDH2 are one of the commonly found in leukemia cells [2]. IDH1 and IDH2 are encoding isomers of isocitrate dehydrogenase – enzyme catalyzing oxidative decarboxylation of isocitrate to α-ketoglutaric acid. Due to enzymes mutations, 2-hydroxyglutarate – a compound with a similar structure to α-ketoglutarate accumulates in cancer cells leading to impairments of histone demethylation [3-4] and to interruption of gene expression. As a consequence, IDH1/2 mutations cause epigenetic and metabolic changes that support AML cell growth [2]. The most common mutations are IDH1/R132H, IDH2/R140Q, and IDH2/R172K [4].

Herein, we used IDH2 mutants, obtained via transfection of IDH2 wild-type-expressing cell line (human erythroleukemia cell line, HEL) to identify spectroscopic signature of IDH gene-driven molecular changes [6].

IDH2 mutant-expressing AML cells were generated by transfection of HEL cells with expression constructs for IDH2-R140Q and R172K. Raman hyperspectral images were acquired with the use of confocal Raman spectrometer WITec Alpha 300R. Raman images were analyzed using k-means cluster analysis (KMCA) and obtained averaged spectra of cells and their components were later subjected to principal components analysis (PCA) and partial least squares regression (PLS).

Our results show that IDH2 mutations, especially IDH2/R172K, cause subtle but significant differences in HEL cells’ biochemical profile. It proves the potential of Raman spectroscopy in the screening of leukemia and studying point-mutations in cancer cells.  The Raman-based identification of IDH mutations might be utilized to reduce the time of diagnosis and could help with the quick introduction of the most efficient treatment regimen to patients.

References

[1] J. L. Liesveld et al., and M. A. Lichtman, in Wiliams Hematology, 9th ed., K. Kaushansky et al, Eds. Nowy Jork: McGraw Hill Education, 2016, 1373.

[2] C. Lu et al., Nature, 483 (7390), 474–478, 2012.

[3] H. R. Madala et al., Cancers, 10 (2), 2018.                      

[4] H. Yang et al., Clinical Cancer Research, 18 (2), 5562–5571, 2012.

[5] F. P. S. Santos et al., Leukemia, 23 (12), 2275–2280, 2009.

[6] P. Martin et al., Science, 216 (4551), 1233–1235, 1982.

Keywords: Raman spectroscopy, IDH mutation, leukemia, chemometrics

Acknowledgments

The „Label-free and rapid optical imaging, detection and sorting of leukemia cells” project is carried out within the Team-Net program of the Foundation for Polish Science co-financed by the EU under the ERDF.

Form: abstract publication