Page 67 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

SDIRSACR Oncology Insights

cystatin F by NK cells leads to a marked reduction in their cytotoxic function by decreasing the activity of effector
molecules granzymes A and B. Similar efficient uptake of extracellular monomeric and dimeric cystatin F has been
observed in primary CTLs and the CD8⁺ T cell line TALL-104. Once internalized, cystatin F localizes to cytotoxic granules,
where it co-localizes with granzyme A and perforin, and its primary binding partner has been identified as cathepsin C.
This interaction ultimately results in reduced cytotoxic activity in both primary human CTLs and TALL-104 cells.
Material and Methods: To comprehensively investigate the role of cystatin F in the context of immune checkpoint
inhibitor (ICI) therapy, we analyzed its expression in cytotoxic T lymphocytes (CTLs) derived from peripheral blood
mononuclear cells (PBMCs) and evaluated its presence in tumor tissues collected prior to treatment initiation. Tumor
samples were used to examine the localization and abundance of cystatin F-expressing cells and to explore potential
relationships with the immune microenvironment. In parallel, we conducted an in silico analysis of publicly available
transcriptomic datasets to assess cystatin F expression patterns across tumor types, its prognostic significance, and its
correlation with immune cell infiltration signatures. Peripheral blood samples were collected from melanoma patients
undergoing first-line ICI therapy—nivolumab, ipilimumab/nivolumab, or pembrolizumab—at the Institute of Oncology
Ljubljana, Slovenia. Samples were obtained at three defined timepoints: 4 weeks prior to therapy, and 12 ± 2 and
28 ± 2 weeks following treatment initiation. Longitudinal profiling of cystatin F expression in CTLs was performed at
each timepoint to capture dynamic changes during treatment. Patients were clinically monitored for up to 12 months
post-treatment initiation and stratified into responder and non-responder groups based on RECIST criteria and overall
clinical outcome. PBMCs were isolated by density gradient centrifugation, cryopreserved, and subsequently thawed
for downstream analysis. CD8⁺ T cells were enriched using magnetic-activated cell sorting (MACS). Flow cytometry was
employed to quantify intracellular cystatin F protein expression in CTLs, while quantitative real-time PCR (qRT-PCR) was
used to measure transcript levels of cystatin F and cytolytic effector genes including perforin and granzymes A and B.
In addition, SDS-PAGE and western blot analyses were conducted to assess the expression and activation state of key
cytotoxic granule components. This multimodal approach enabled us to link molecular and functional profiles of CTLs
with clinical response to ICI therapy and to evaluate the utility of cystatin F as a predictive biomarker of therapeutic
efficacy.
Results and Conclusions: Our longitudinal analysis demonstrated that cystatin F expression in cytotoxic T lymphocytes
(CTLs) declined progressively in melanoma patients who responded to immune checkpoint inhibitor (ICI) therapy. This
temporal decrease was most pronounced in individuals achieving durable clinical responses, suggesting that elevated
cystatin F expression may be associated with impaired CTL function, while its downregulation could reflect restored
or enhanced cytolytic activity during effective treatment. These findings support the potential role of cystatin F as a
dynamic biomarker of immunotherapy response, particularly in the context of CD8⁺ T cell-mediated tumor clearance.
Although subtle differences in cystatin F kinetics were noted between male and female patients, these observations
require validation in larger cohorts and were not the primary focus of this investigation. Taken together, our results
underscore the clinical relevance of monitoring cystatin F expression in peripheral CTLs during the course of ICI therapy.
As a regulator of effector function in cytotoxic immune cells, cystatin F may serve as a useful indicator of therapeutic
efficacy and disease trajectory in melanoma patients. Incorporating cystatin F into existing biomarker panels could
enhance patient stratification and aid in tailoring treatment strategies, thereby improving outcomes while minimizing
unnecessary exposure to ineffective therapies. Future studies should focus on validating these findings across broader
patient populations and further elucidating the molecular mechanisms underlying cystatin F modulation in the setting
of immune checkpoint blockade.

Acknowledgments and funding: Slovenian Research Agency (ARIS): P4-0127 (to JK) and P3-0321 (to JO).

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