METACELL. PERSONALIZED CANCER THERAPY USING CIRCULATING TUMOR CELLS (CTCs) METACELL LIQUID BIOPSY

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1 METACELL PERSONALIZED CANCER THERAPY USING CIRCULATING TUMOR CELLS (s) AN EASY WAY TO LIQUID BIOPSY MORE THAN A METASTATIC CELL IN BLOOD A STEP TOWARDS PERSONALIZED CANCER TREATMENT LIQUID BIOPSY REAL-TIME INFORMATION ON TUMOR PROGRESSION COST-EFFECTIVE WAY TO TAILOR THE THERAPY INCREASE OF THERAPY EFFICIENCY REDUCTION OF DRUG SIDE-EFFECTS METACELL EASY, FAST AND INEXPENSIVE NO EXTRA COSTS ALL TYPES OF SOLID TUMORS - CELL SURFACE ANTIGEN-INDEPENDENT HUMAN AND ANIMAL SAMPLES GENTLE TO ISOLATED CELLS KEEPING s AND DTCs VIABLE FOR CULTIVATION AND DOWNSTREAM ANALYSIS

2 The MetaCell technology SIMPLE: MetaCell uses a capillary action driven size-based separation of rare blood cells from the unclotted peripheral blood. The MetaCell enables separation and detection of rare cells within a single step. Approximately 8-10 ml of blood is passed through a MetaCell tube; the collection of the s/dtcs on the separating membrane takes 2 minutes. MetaCell technology does not require any expensive equipment or additional investment. One test = one tube. Please add remark on microscope. SENSITIVE: MetaCell is size-based specific to rare cells in peripheral blood without dependency on the tumor cell membrane antigens (EpCAM, HER2, MUC1, EGFR, etc.). MetaCell technology is suitable for enrichment of rare cells spread from epithelial as well as mesenchymal solid tumors. Separation of circulating endometrial cells from blood of endometriosis patients is also effective. SOFT: MetaCell is soft to the separated cells, keeping them viable. After the separation, the viable intact cells are suitable for subsequent characterization and/or in vitro cultivation. The cell separation and cultivation platform combines sophisticated size-based separation with cultivation on the membrane principle.

3 Features Cat. No.: EMC001 Packaging: 10 pcs/box Enrichment of intact of s/dtcs/ circulating endometrial cells (CECs)/ circulating tumor microemboli (CTM) in very short time (2-3 min) No problems with flow-based blood coagulation Isolation of both epithelial and mesenchymal tumor cells without use of antibodies against cell membrane antigens High detection capability 1 tumor cell in 8-10 ml of blood Separation process does not require any use of lysis reagents: (a simple gentle filtration without affecting the cell character) Preservation of the morphological attributes of isolated cells Possibility to count isolated cells immediately after separation process Possibility to culture enriched cells in vitro for subsequent use/analysis/ characterization No need for accessory equipment/ machine for cell separation, no space requirements Applications The MetaCell technology enables capture culture characterization The MetaCell technology can be used in Science: Principles of cancer dissemination may be observed in parallel to the primary tumor tissue and metastasis Health-care providers: Personalized therapy within precision medicine could save patients from side effects and enormous treatment costs. Detailed information on the characteristics cells enriched from circulation or body fluids enables the clinicians to select an appropriate treatment for their patients. Pharma and biotech industry: Co-development of drug and companion diagnostics, discovery of new druggable markers (s offer a new possible target for oncotherapy) The use of the MetaCell tests has been proved in Oncology: Breast cancer, gastric cancer, lung cancer, esophageal cancer, pancreatic cancer, prostate cancer, urothelial tumors Gynecology: Circulating tumor cells (s), circulating endometrial like cells (CECs), circulating trophoblasts (CTLC), breast cancer, ovarian cancer, cervical cancer, endometrial cancer, endometriosis, pregnancy Animal studies: Orthotopic metastatic nude mice models (Human cancer lines are implanted in nude mice. Please see the references list.)

4 Identification Process Cytomorphology + Molecular Diagnostics = Protocol 30 min Blood collection: Approximately 8-10 ml of venous blood is drawn from the antecubital veins and placed into an EDTA-containing tube. Size-based cell separation: 8 ml of peripheral blood sample (can be extended up to 50 ml) is transferred into the tube and filtered through an 8 μm-pore polycarbonate membrane. In vitro culture set up and cytomorphological analysis: The membrane is transferred into a 6-well cultivation plate and observed by inverted microscope. Cytomorphology Filtration In vitro culture set-up Microscope: In vitro cytomorphology evaluation using vital fluorescence stains Cell plasticity and deformability (Bottom-well cell fraction) % of cells on the membrane (Membrane cell fraction) Molecular Diagnostics Mutational analysis Methylation analysis DNA RNA Gene expression profiling Tumor associated markers Therapy-related markers (chemoresistance, targeted therapy) s and DTCs cultivation and downstream analysis: Separated cells can be cultured and the grown cells can be analyzed by histochemistry (e.g. MGG staining) immunohistochemistry using specific antibodies to determine the cell origin (membrane is compatible with staining machines e.g. Ventana) vital fluorescence staining, which enables to use stained cells for further downstream analysis can be used gene expression profiling (mutational analysis, methylation analysis, tumor associated markers, therapy-related markers).

5 Clinical indications for examination / Breast cancer study Circulating tumor cell () examination was indicated as a complementary test during neoadjuvant therapy in 20 patients with breast cancer. The presented findings support the use of liquid biopsy to enhance monitoring of malignant disease and prediction of treatment efficacy. During the -examination presence was monitored. In parallel, gene expression analysis was performed on isolated enriched s. Tumor associated genes and genes associated with chemoresistance were evaluated in comparison to the administered therapy (anthracyclines/ taxanes/ anti-her2 therapy). In general, elevated -count, indicates chemoresistance, usually. However, timely retuning of therapy based on -chemoresistance data obtained with the liquid biopsy resulted in reasonable decreases in tumor progression expressed as the -count. DG. NEOADJUVANT CHEMOTHERAPY SURGERY ADJUVANT TR. ANTHRACYCLINES ANTHRACYCLINES amount CHEMORESISTANCE CHEMORESISTANCE MRP1 DG. MRP1 MRP2 NEODAJUVAT CHEMOTHERAPY ANTHRACYCLINES CHEMORESISTANCE MRP1 MRP2 SURGERY ADJUVANT TR. TAXANES DG. TIME MRP1 MRP2 NEODAJUVAT CHEMOTHERAPY TAXANES SURGERY What if the chemosensitivity test is performed before chemotherapy start? ADJUVANT TR. TAXANES CHEMORESISTANCE MRP1 SITUATION 2: During the AC treatment the therapy is changed (based on resistance) to taxanes. The tumor is reduced and the number of s decreases. After the surgical removal, the tumor is reduced and s decreases. CHEMORESISTANCE CHEMORESISTANCE MRP1 TIME SITUATION 1: The standard neoadjuvant with anthracyclines (AC). Regardless of whether the tumor is reduced, the number of AC resistant s increases (increased expression of MRP1); the treatment continues. The patient is indicated for surgical removal of the tumor. After tumor removal, the number of s decreases for a while but then again rises with the formation of metastatic lesions. TIME SITUATION 3: At the very beginning of the treatment, presence of AC resistance on s is determined, therefore the taxanes are indicated immediately. After the therapy start, tumor volume is reduced and the number of decreases.

6 References Kolostova, Katarina, et al. The added value of circulating tumor cells examination in ovarian cancer staging. American journal of cancer research 5.11 (2015): Kolostova, Katarina, et al. Molecular characterization of circulating tumor cells in ovarian cancer. American journal of cancer research 6.5 (2016): 973. Kolostova, Katarina, et al. Isolation, primary culture, morphological and molecular characterization of circulating tumor cells in gynecological cancers. American journal of translational research 7.7 (2015): Kolostova, Katarina, et al. Morphological and gene-expression characterization of viable heterogeneous circulating tumor cells size-captured and cultured from triple-negative breast cancer mouse models. Int J Clin Exp Med 9.5 (2016): Kolostova, Katarina, et al. In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein. Journal of fluorescence 24.5 (2014): Bobek, Vladimir, Katarina Kolostova, and Eduard Kucera. Circulating endometrial cells in peripheral blood. European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014): Bobek, Vladimir, et al. Cultivation of circulating tumor cells in esophageal cancer. Folia Histochem Cytobiol 52.3 (2014): Kolostova, Katarina, et al. Detection and cultivation of circulating tumor cells in gastric cancer. Cytotechnology 68.4 (2016): Bobek, Vladimir, et al. Circulating tumor cells in pancreatic cancer patients: enrichment and cultivation. World Journal of Gastroenterology: WJG (2014): Bobek, Vladimir, Martin Cegan, and Katarina Kolostova. Circulating tumour cells in patients with urothelial tumours: Enrichment and in vitro culture. Canadian Urological Association Journal (2014): Distributed by Manufactured by BioVendor GesmbH Gaudenzdorfer Gürtel Vienna, Austria Phone: Fax: infoaustria@biovendor.com BioVendor GmbH Otto-Hahn-Straße Kassel, Germany Phone: Fax: infoeu@biovendor.com Oxford Biosystems Ltd 115J Olympic Avenue Milton Park, Oxfordshire OX14 4SA, United Kingdom Phone: sales@oxfordbiosystems.com MetaCell s.r.o. Erbenova 783/ Ostrava, Czech Republic info@metacell.cz