Summary and Conclusions
CApaCIty
The CAPACITY project revolved around the study of immune receptors present in cancer stem cells as therapeutic targets in skin squamous cell carcinoma.
Skin squamous cell carcinoma is one of the most common cancer worldwide. Currently, the treatment options, surgery and radiotherapy procedures are associated with significant patient morbidity, discomfort, and undesirable scarring. On the other hand, the systemic pharmacological treatment available has several side effects, therefore are not a preferred option. The development of a new specific treatment is therefore urgent to treat these patients.
Cancer stem cells initiate, sustain the prevalence, and resistance to therapy of skin squamous cell carcinomas. This is a complex process dependent on genetic modification and more interesting determined by the interaction between these cancer cells and the cells present in the tumor microenvironment, in particular macrophages cells. Therefore, the use of therapies able to block these interactions is a promising advance to eradicate skin squamous cell carcinoma.
Within the CAPACITY project, using a comprehensive and integrated approach based on human tissues, cell biology, medical chemistry, and advanced technologies, I set the ambitious aim to understand and reveal the role of immune receptors present in cancer stem cells that can be used as therapeutic targets in skin squamous cell carcinoma.
The objectives of this Marie Skłodowska Curie Action (MSCA) have been to characterize the presence and the functions of relevant immune receptors in the cancer stem cells in human skin squamous cell carcinomas; 2) Analyzed the relevance of immune receptors as therapeutic targets using new peptide-based inhibitors of protein-protein interactions.
The project results revealed that CD99, a membrane protein present in cancer stem cells in skin squamous carcinoma have an impact on the cancer stem cells characteristics and in their connection with macrophages. The analyzed data resulted in new information regarding genes and mechanisms implicated in skin squamous cell cancer. Moreover, peptide-based inhibitors developed in this project may have the potential used in vivo to block squamous cell carcinoma growth.
Overview of methods and results
To characterize the expression of the immune receptors in different clinic stages of human skin squamous cell carcinoma, immunofluorescence was performed in human paraffine tissues using the identified immune receptors, CD99, MFGE8 or Nectin2 and cancer stem cells markers (SOX2+) (WP1). 196 samples from skin squamous cell carcinoma were used and it was observed high expression in SOX2+ cells and higher expressed in stage II of squamous cell carcinoma and no differences were found by gender or age.
Additionally, the role of these immune receptors in cancer stem cells features and the macrophage interaction was investigated in vitro, using skin squamous cell carcinoma cell lines and genetic loss functions studies (WP2). Once KD/KO cells were achieved differently, in vitro assays were performed to study the effect of their loss of expression in the cell behavior (proliferation, apoptosis, colony formation assay, cell cycle analysis, migration/invasion) and cellular interactions (adhesion assay, proximity assay). These results showed that CD99 protein sustain cancer cells proliferation and it is required for the cancer cell – macrophages interactions in vitro.
To further investigate the mechanisms behind the influence of CD99 in cancer stem cells-macrophage interactions, in vitro systems using CD99 deficient cells/controls and macrophages were developed (WP2). Using DNBseq™(BGI-Shenzhen), 21 transcriptomes were sequenced in total. Gene Set enrichment and gene ontology analysis were performed between the different conditions. The results reveal that multiple molecular components seem to contribute to the intracellular mechanisms involved in the cancer stem cells-macrophage interactions. My data indicate a significant contribution of adhesive and cytoskeleton proteins. Interestingly, my results reveal many differentially expressed transcripts from important signaling pathways involved in cancer. These results indicate that there is a need to experimentally verify the expression of those transcripts and proceed with their functional characterization.
The last part of my CAPACITY project involved the development of peptide-base inhibitors (WP3). Based on the previous results from the project, a blocking peptide was designed and synthesized against CD99 protein. CD99 was cloned and overexpressed in E. coli, and peptide sequences were synthesized by automatized and high-throughput solid-phase peptide chemistry and purified using preparative high-performance liquid chromatography (HPLC). Peptides showed stability for 24h in serum with increased degradation after 72h. The efficiency of peptide inhibition was determined in vitro by competition assays and by using squamous cell carcinoma cell lines. The peptides showed specify through CD99 and reveal inhibition of cancer stem cells-macrophage interactions in vitro. These results indicate that there is a need to further characterized these peptide-based inhibitors in vivo to analyze their potential as a therapeutic drug against skin cancer.
Potential impacts of the project
The work carried out within the CAPACITY project has resulted in deepening the knowledge and understanding of skin squamous cell carcinoma. The comparative transcriptomics on the comprehensive experimental set-up provided new information regarding a) important genes and pathways downstream to CD99 involved in cancer stem cells features and b) the gene regulation during cancer stem cells-macrophage interaction. Among the novel results of the CAPACITY project is the development of peptide-inhibitors of protein-protein interactions. Using these peptides, we observed that CD99 has high potential effectiveness as an immunotherapeutic target against human skin squamous cell carcinoma.
The overall results present a lot of potential of high impact for biomedical sciences. Understanding the mechanisms and identifying the molecules implicated in skin squamous cell carcinoma is fundamental for improving the prevention and the development of new therapeutic methods against this cancer.