Page 91 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

SDIRSACR Oncology Insights

L32

Tracking Osteosarcoma Progression: A Longitudinal Single-Patient Study Integrating Functional Analyses
and Single-Cell Transcriptomics

Karla Ostojic¹*, Paula Stancl¹*, Rosa Karlic¹, Marina Panek² ,Ursula Prosenc Zmrzljak³, Marijana Simic Jovicic⁴, Katarina
Caput Mihalic¹, Lucija Šerić Jelaska¹, Jakob Šimić¹, Inga Urlic¹

1 University of Zagreb, Faculty of Science, Zagreb, Croatia
2 Labena d.o.o., Zagreb, Croatia
3 BIA Separations CRO, Ljubljana, Slovenia
4 Children’s Hospital Zagreb, Zagreb, Croatia

Keywords: cancer stem cells, chemotherapy resistance, telomere extension, osteosarcoma, single-cell RNA sequencing

Background: Osteosarcoma (OS) is the most common malignant bone tumor, peaking in adolescence and later in
adulthood. Despite defined histological subtypes and grading, predicting disease progression remains challenging due
to its rarity, genetic complexity, and heterogeneity. While most cases arise sporadically, some are linked to inherited
syndromes such as Li-Fraumeni syndrome or retinoblastoma. Advances in chemotherapy have improved survival
for localized OS to over 65%, yet outcomes for metastatic disease remain poor. Tumor progression and recurrence
are driven by accumulating mutations and selective pressures[1] , leading to therapy-resistant, highly adaptable cell
populations. A deeper molecular understanding is crucial for improving prognostic and therapeutic strategies.
To better understand these dynamics, we conducted a longitudinal study tracking tumor evolution in a single patient
across three key clinical stages: diagnosis, after neoadjuvant chemotherapy, and recurrence. The patient, a young male
diagnosed with grade 3 chondroblastic OS of the femur, was treated with the EURAMOS-1 chemotherapy protocol [2]
followed by surgical resection and additional adjuvant therapy. Upon recurrence, a second surgery was performed.
Tumor samples were collected at each stage, and primary cell cultures were established and enriched for cancer
stem cells (CSCs). These cultures were then evaluated using a multimodal platform that included morphological and
biophysical analyses, functional assays, and single-cell RNA sequencing (scRNA-seq).
Materials and Methods:
Patient Samples and Ethical Approval
Tumor material was obtained from a male patient with grade 3 chondroblastic osteosarcoma of the femur at three
time points: diagnostic biopsy, post-neoadjuvant chemotherapy resection, and resection following recurrence. All
samples were collected at the Children’s Hospital in Zagreb with ethics committee approval and informed consent.
U2OS (osteosarcoma) and HeLa (cervical carcinoma) cell lines were used as experimental controls.
Primary Cell Culture and CSC Enrichment
Primary cell cultures were established from all tumor samples. Cancer stem cells (CSCs) were enriched using spheroid
culture in serum-free DMEM/F-12 medium supplemented with FGF2, EGF, ITS, ammonium bicarbonate, progesterone,
putrescine, and 2% methylcellulose on low-attachment plates. Adherent 2D cultures were maintained in DMEM/F-12
with 10% fetal bovine serum (FBS) and antibiotics. Cells were passaged at 80–90% confluency and used between
passages 3 and 7.
Spheroid Formation Assay and Imaging
To quantify CSC frequency, monolayer cells were dissociated into single-cell suspensions and seeded into low-
attachment 96-well plates at a density of 10 cells/μL. Spheroids were cultured for seven days and analyzed by inverted
microscopy. Spheroid-forming efficiency (SFE) was calculated as:
SFE (%) = (number of spheroids formed / number of seeded cells) × 100.
Adherent cell morphology was evaluated using the HoloMonitor® Live Cell Imaging System over 48 hours. Monitored
parameters included optical thickness, shape irregularity, and perimeter.
Spheroid properties (mass density, diameter, and weight) were assessed with the W8 device (CellDynamics) based on
sedimentation analysis [3]. Spheroids were grown for 10 days, fixed in 4% PFA, and analyzed in duplicate. Statistical
evaluation was performed using Student’s t-test.
3D hydrogel-based spheroid imaging was performed using tyramine-conjugated gelatin and hyaluronic acid hydrogels,
crosslinked with HRP and H₂O₂. Spheroids were imaged every 30 minutes for seven days. Differences between conditions
were analyzed using the Kruskal–Wallis test followed by the Bonferroni-corrected Dunn’s post hoc test.
Migration Assay
To assess migratory potential, a wound healing assay was performed. Once cell confluence was reached in 6-well plates,
a scratch was made, and cell migration was monitored every 3 hours for 48 hours using HoloMonitor imaging. Cells
were maintained in serum-free medium to suppress proliferation. Migration was quantified using dedicated software.

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