Harnessing the tumor draining lymph node for effective anti-tumor immunity in breast cancer and melanoma

Part 1: Technical aspects of TDLN immune monitoring Viable cell scrapings of the surgically retrieved and bisected SLN offer a unique opportunity to study tumor induced and/or immune modulator effects. In chapter 2, as part of a special issue on new guidelines for DC research for the European Journal of Immunology (submitted), we describe our method of obtaining viable LN cell suspensions and their use for flowcytomety based characterization of TDLN DC subsets. Cryopreservation of SLN single-cell suspensions allows for simultaneous phenotypic multi-parameter analyses and minimizes operator dependent variability. This would be of particular importance for immune monitoring in future multi-center trials. In Chapter 3, we tested the feasibility of cryopreservation of viable SLN cell samples for flowcytometric analysis, by comparing quantitative DC and T Cell analyses of SLN cell samples after freeze-thawing with direct analysis of fresh SLN cell samples (n=9). Although these techniques should not be employed interchangeably in the same trial, cryopreservation and thawing is a valid alternative to direct analysis of fresh viable lymph node cells, without introducing cryo-dependent variance between SLN samples. Part 2: Breast cancer In order to unravel the immune landscape in BrC TDLN, in Chapter 4, we describe the results of a comprehensive flow cytometry–assisted study of viable cells from BrC SLN (n=58) in a comparative analysis with healthy (i.e. prophylactic mastectomy-derived) axillary lymph nodes (HLN, n=17) and show that BrC induced immune suppression coincides with selectively hampered activation of LNR DC subsets. The knowledge that CpG-B can preferentially activate these LNR DC subsets but also induce STAT-3 (a known poor prognostic indicator in BrC and the master switch of tumor induced immune suppression) prompted the ex-vivo functional validation of combined TLR9 targeting and JAK2/STAT3 inhibition as a potential treatment strategy in breast cancer patients. In Chapter 5 Immune modulatory effects of CpG-B with or without the JAK2/STAT3 inhibitor AG490 are assessed in ex vivo cultured BrC SLN-derived single-cell suspensions (N=26). Although CpG-B alone induced activation of all DC subsets, combined inhibition of the JAK2/STAT3 pathway resulted in superior DC maturation and most profoundly so for LNR DC subsets. Most importantly, combined CpG-B and JAK2/STAT3 inhibition (but not CpG-B alone) significantly enhanced BrC specific T cell reactivity. Part 3: melanoma In this part of the thesis we aimed to investigate the involvement of TDLN in the biological activity of CTLA-4 inhibitors in melanoma. In chapter 6, the clinical safety and tolerability and immunological effects of a single dose of anti-CTLA-4 (tremelimumab) injected intradermally at the tumor excision site (for optimal TDLN access) 1 week prior to SLNB in patients with early-stage melanoma (n=13) are reported. Critical findings in this study underscore the importance of immune modulation of TDLN in generating de novo as well as boosting “dormant“ anti-tumor T cell responses and support the concept of local instead of systemic anti-CTLA-4 blockade. In chapter 7, we review the results of recent (pre)clinical studies pointing to the key role of TDLN in the efficacy of immune checkpoint blockade (ICB) further discuss the evidence that therapeutic targeting of TDLN may ensure sufficient antitumor T cell activation and subsequent tumor infiltration to facilitate effective ICB. In Chapter 8 the studies described in this thesis are summarized and further discussed and in Chapter 9 the main findings of these studies are integrated to highlight clinical implications and future directions in breast cancer and melanoma immunotherapy.