Local immune response in cutaneous basal cell carcinoma

Abstract

BCC is an immunogenic tumor highlighted by the increased risk in immunosuppressed individuals and the frequent occurrence of tumor infiltrating lymphocytes (TILs) in the tumor surroundings. Immunotherapy is evolving as a promising treatment strategy for several cancer types where topical immunostimulators are among the possibilities for superficial BCC. The overall aim of this thesis is to characterize the immunologic response upon BCC as well as characterizing the surrounding tumor stroma. The aim was achieved by the use of a variety of laboratory techniques; immunohistochemistry, immunoflourescence, qRT-PCR and NGS. Tumor microenvironment: T-regs are a subpopulation of the CD4 positive T-cells normally comprising around 5-10% of the peripheral T-cells and up to 20% of the skin resident T-cells. In the healthy individual they are crucial in hindering autoimmune diseases whereas the role in cancer is less advantageous with association to tumor progression for a variety of cancer types. By investigating the presence of T-regs in BCC by immunohistochemistry in study I, it was found that T-regs comprised 45% in mean of the total CD4 positive cells in BCC. The increased T-reg concentration was confirmed with qRT-PCR showing increased Foxp3 expression levels in BCC as well as in the peritumoral skin. In the normal non-UV exposed buttock skin, no Foxp3 expression was found. Hence, T-regs seem to play a role both in BCC but also in the tumor surroundings. Tumor surroundings are essential in terms of the ability for a tumor to grow. Apart from interaction between immune and cancer cells, also crosstalk with cells of the connective tissue such as CAFs is essential. In study II, NGS revealed increased expression of the CAF-markers P4H and PDGFR-β in BCC. Subsequent qRT-PCR confirmed this and also showed increased expression in the peritumoral skin whereas no expression was found in the normal buttock skin. FAP-α expression was seen only within BCC. CAFs are thus highly present within BCC and we further hypothesize that fibroblasts in the peritumoral skin acquire a phenotype intermediate between normal fibroblasts and CAFs in BCC. This intermediate phenotype might be induced by chronic UV-exposure mediated by increased IL6 expression. This corresponds to our findings of highly increased IL6 expression primarily in the peritumoral skin and to previous literature describing CAF-induced tumor-promoting IL6 expression upon UV-exposure in cutaneous SCC. Recruitment of TILs to BCC: mRNA expression levels of the chemokines CCL17, CCL18, CCL22 and CXCL12, involved in T-reg attraction to tumor sites were increased both in tumor and peritumoral skin with lack of expression in the normal skin. Correlation between the chemokines CCL17 and CXCL12 and CAF markers was found by IF establishing a role for CAFs in attracting T-regs to tumor sites. Efficient immunologic anti-tumor response could be provided by clonal expansion of T-cells directed against tumor-antigens. If this was the case, then restricted TCR-repertoire in BCC compared with surrounding skin would be seen. Analysis of the α and β- chain of the TCR was performed showing a high diversity of TCR repertoire in BCC and lack of predominant V(D)J-gene usage, no preferential VJ pairing or specific CDR3 length distribution. Therefore, no support of antigen-driven clone selection was found. This corresponds with lack of obvious anti-tumor skewing towards a Th1, Th2 or Th17 polarization.

Conclusion: To summarize it has been shown, with these studies on the local immune response upon BCC development, that an immunologic response is generated in line with BCC being an immunogenic tumor. This response is not specific, though. Additionally, BCC is capable of generating a protective niche in the microenvironment composed of both T-regs and CAFs breeding local immunosuppression and hindering of adequate anti-tumor response. In a clinical perspective, further research in improving immunotherapy for BCC is promising since an immunological response is present but needs to be reactivated.