Investigation and discovery of targetable markers in urological tumors

Abstract

Urological cancers are among the cancers with the highest incidence. In recent years, several therapeutic solutions have been suggested and accepted in the clinic for treatment, but tumors, especially the most aggressive forms, frequently develop resistance to the available therapies. Thus, we focused on investigating novel solutions for treating the most aggressive forms of urological cancers. Specifically, we aimed to:<br /> 1) Investigate the application of nanobodies (Nbs) as targeting agents for CUB domain-containing protein 1 (CDCP1) in prostate cancer (PCa) and epithelial growth factor receptor (EGFR) in the basal/squamous (Ba/Sq) subtype of bladder cancer (BCa).<br /> 2) Study the correlation of CDCP1 with tumor-associated macrophage (TAM) infiltration in PCa and BCa.<br /> 3) Characterize senescence for the discovery of new senescence markers in advanced PCa.<br /> CDCP1 is a transmembrane protein that correlates with tumor stage in several cancers. In our experience, CDCP1 is a promising target for treating advanced PCa. Thus, we tested Nbs as an innovative strategy for its targeting. Nbs are the smallest existing region of an antibody (Ab) that maintains the binding to their target and have several advantages compared to Abs. They are 10 times smaller, making them ideal for treating solid tumors since they can diffuse better in the tumor mass. Moreover, they are characterized by cheaper and easier production and higher stability. We also considered the application of Nbs for targeting EGFR, a transmembrane receptor known for sustaining tumor growth. Drugs targeting EGFR are already used in lung and head and neck cancers. In this work, we investigated EGFR targeting in the Ba/Sq subtype of BCa. This subtype is characterized by high levels of EGFR and greater aggressiveness. Both targeting CDCP1 and EGFR with Nbs in these cancers revealed promising results. Indeed, Nb dimers targeting CDCP1 reduced tumor growth in a PCa cell model, and Nbs targeting EGFR reduced cell growth in a Ba/Sq BCa cell model.<br /> In the second part of this work, we studied the correlation of CDCP1 with TAM infiltration. CDCP1 correlates with the Ba/Sq subtype of BCa which is typically infiltrated by immune cells. Our results showed that CDCP1-expressing tumor cells produce high levels of CCL2 and IL6, contributing to macrophage recruitment. Macrophages infiltrating CDCP1 tumors in our PCa mouse model overexpressing CDCP1 have the typical characteristics of tumor-associated macrophages (TAMs), which are reported to create an environment that promotes tumor growth and reduces immune responses. Thus, their elimination from the tumor microenvironment (TME) proves essential to dampen tumor aggressiveness and improve tumor responsiveness to immune therapy. Treatment with mAbs targeting CDCP1 efficiently reduced CCL2 and IL6 production and the migration potential of macrophages.<br /> In the final part of this work, we aimed to discover new senescence markers to combine chemotherapy, such as docetaxel, with more efficient senolytic drugs. This proves fundamental since senescent cells could cause tumor relapse or aggravate tumor growth due to the production of growth factors and cytokines. However, targeting senescent cells is challenging due to their similarities with normal cells. To find a suitable marker for senescent cell elimination, we first developed a new model for senescence individuation using the CRISPaint technology. This technology allowed for the visualization of senescent cells in real-time and suggested that the situation after treatment with docetaxel in PCa is more complicated than expected. Indeed, we did not observe the formation of a senescent population but the presence of 3 different populations. We separated these populations and performed mRNA sequencing to identify specific population markers. We obtained a set of 8 genes that are progressively up- or down-regulated along the 3 populations, suggesting that different levels of senescence characterize these populations. To investigate this further, we plan to perform single-cell sequencing.<br /> Altogether, the results presented in this thesis suggest some solutions for treating aggressive BCa or PCa. In particular, using Nbs to target CDCP1 or EGFR and targeting CDCP1 to reduce TAM infiltration. Finally, this thesis reveals the complexity of the senescent phenotype that requires a deeper investigation and an improved definition before being targeted efficiently.