Michal Masařík Research Group

Role of cancer-associated fibroblasts

In our lab, we focus on the process by which tumour cells reprogramme their microenvironment (TME), including changes in intercellular communication and the alterations in the metabolic phenotype of TME cells. Fibroblasts are an essential component of the TME and reflect stress in the TME. Such stress responses in fibroblasts can include their activation into cancer-associated fibroblasts (CAFs). The pleiotropic effect of CAFs in the TME is likely to reflect the heterogeneity and plasticity of their population, with context-dependent effects on carcinogenesis. CAFs provide many targetable molecules that could play an important role in future cancer therapy.

Role of microbiome

The TME is a complex environment that includes not only human cells but also the community of microorganisms known as the microbiome, including viruses, mycoplasmas, bacteria, archaea and bacteriophages. The human microbiome is a complex ecosystem that interacts closely with the human host. Residues of these microbes, such as DNA, RNA, peptides and cell wall components, have been detected in cancer cells and tumour-infiltrating immune cells. This finding suggests that microbial elements can directly interact with the cellular machinery of both cancer and TME cells, potentially influencing their behaviour and functions. We focus on the investigation of the complex interplay between genetics and environmental factors mediated by the microbiome in heterogeneous cancers that lack strong driver mutations with high penetrance.

Extracellular vesicles (EVs) are membrane-derived vesicles released by cells into the extracellular space and play an important role in cell-cell communication and the regulation of many biological processes. Similar to apoptotic cells, the major type of EVs released by cancer cells and CAFs display phosphatidylserine (PS) on their outer membrane layer. Phosphatidylserine-positive EVs (PS+EVs) can prepare the soil for tumour seeding and create a suitable TME. These vesicles can not only promote cancer cell proliferation and metastasis but also facilitate drug resistance of cancer cells and even orchestrate crosstalk between tumours and neighbouring cells, such as cancer-associated fibroblasts (CAFs).

Our research focuses on changes in the protein and RNA content of PS+EVs isolated from CAFs and HNSCC cells under normal and stress conditions (e.g. lysosomal dysfunction, starvation). We are also investigating the impact of these EVs on the biological response of recipient cells, such as metabolic changes, migration, or sensitivity to cell death, during crosstalk between HNSCC and CAFs. Our work aims to better understand the role of PS+EVs in intercellular communication in the HNSCC microenvironment and to identify potential biomarkers indicative of the processes involved in tumour progression

We are members of the International Society for Extracellular Vesicles (ISEV) and co-founders of Czech Society for Extracellular Vesicles (CzeSEV).

The mechanical properties of cells are affected by the cell cycle, differentiation, and pathological processes such as malignant transformation, cardiovascular disease or ageing. The ability of cancer cells to deform is related to their ability to spread - most tumour cells are typically softer than their non-tumour counterparts. Although the main determinants of the mechanical properties of cells are known, there are still unanswered questions in mechanobiology such as how intracellular architecture, cell metabolism and their changes relate to the ability of cells to migrate and invade. These are the questions we are focusing on in our laboratory. Our long-term goal is to translate this knowledge to the bedside to improve cancer diagnosis. To this end, we are also developing methodological approaches to quantify cell viscoelasticity using quantitative phase microscopy and shear stress-induced cell deformation, where we are able to measure hundreds of cells per minute - orders of magnitude more than conventional atomic force microscopy.

In addition to routine cell biology-oriented experiments in the lab, we employ the following techniques.

Quantitative phase imaging

Quantitative phase imaging, a microscopic technique, offers valuable insights into cellular dynamics by providing quantitative measurements of light phase changes within living cells. This non-invasive approach enables precise label-free time-lapse determination of cell dry mass or refractive index, thereby characterization of cellular processes such as cell growth, or response to the treatment of the cell. These changes can be observed without fixation and labelling which severely change cell characteristics as fluorescent labelling can affect cell behaviour during long experiments due to phototoxicity.

We employ this technique for characterisation of analysis of growth and migration of populations of cells, for analysis of biophysical properties of cells following various stimuli, and for quantification of cell death subroutines.

Extracellular vesicle isolation and functional studies

We have successfully optimized the methodology for isolation of extracellular vesicles, their characterization and testing of their effect on recipient cells. We can isolate EVs from both conditioned media and various body fluids such as blood serum.

Patient-derived primary cultures and patient-derived xenografts

Compared to stable cell lines, from tumour tissue samples we can isolate primary cultures of both tumour and stromal cells and provide valuable associated information about patient prognosis. In our laboratory, we use these approaches extensively to characterize the transcriptome of tumour cells and to promote various subclones, particularly cancer-associated fibroblasts, allowing us to monitor in vitro, how CAFs derived from specific patients affect tumour cell behaviour.

In vivo models

In our laboratory, in vivo experiments are conducted using model organisms to investigate different aspects of cancer biology, the effects of therapeutic interventions, and the toxicity of novel anticancer compounds. Our experiments involve orthotopic and heterotopic applications of cancer cells to analyse tumour growth and dissemination. A key focus of our in vivo experiments is the analysis of lung metastatic foci, providing insights into cancer metastasis and therapeutic efficacy. Moreover, we utilize these model organisms to assess the toxicity and efficacy of novel anticancer compounds.

Chicken chorioallantoic membrane assay

We also perform a chicken chorioallantoic membrane (CAM) assay which is an intermediate step between in vitro and in vivo experiments, in both in ovo and ex ovo platforms. We can prepare cell lines onplants or patient-drived xenografts (PDX) and angiogenesis assay. We use CAM to evaluate therapeutic interventions, effects on the metastatic potential of cancer cells, toxicity of new anticancer agents or for influencing blood vessel formation.

AI in image processing and data analysis

In line with the acquisition of microscopic image data, our lab employs artificial intelligence (AI) for the advanced analysis of cellular images. This AI-driven approach automates cell image segmentation and analysis, significantly enhancing efficiency and precision. Leveraging machine learning and AI, we can accurately identify and track cellular features, streamlining the study of cell growth, migration, and response to treatments. This method allows for the analysis of large volumes of data, providing insights into cellular dynamics without the need for time-consuming manual steps during analysis.

We combine those AI-driven approaches with traditional methods for image and data analysis for various types of data including images from confocal microscope and quantitative phase imaging. This allows us to automatize cell death type recognition, mechanical properties extraction, automatic analysis of response to treatments, and various other applications.

Our laboratory has developed a QPI-based method to detect cell death and determine its specific subtype (apoptosis or lytic cell death) without the use of dyes, based only on the quantitative phase dynamics of the cell.