Early immunological events in the periphery and the TME following a local immunostimulating instillation into the bladder in the MB49 orthotopic model

The mouse bladder 49 (MB49) syngeneic tumors respond to various immunotherapies, both stimulatory immunotherapies and check-point blockers; when tumors are cured it is often an anti-tumor memory response established (previous publications by our group and others). Herein we present data suggesting that bladders of orthotopic MB49 tumor bearing mice hold a reservoir of lymphocytes surrounding the tumor, wherein they migrate rapidly upon immune adjuvant stimulation. The tumors appear to form an accessible immune microenvironment rich in vessels that mediate the homing of leukocytes to the tumor site. This is in contrast to MB49 tumors that grow subcutaneously and do not appear to provide the same rapid immune cell infiltration capacity. After 24 hours post a bladder restricted TLR9 stimulation, we could identify that CpG ODN 1668-treated tumor-bearing bladders displayed increased vascularization around the tumor. Interestingly it appeared as CCL21 expression intratumorally aligned with increased recruitment of CD11c+ DCs in the tumor TME, rather than a migration to lymph nodes. Intratumorally, CCL21 expression correlated with infiltration of both CD11C and CD8 cells. As CD11c+ dendritic cells migrate towards gradients of CCL21 via their CCR7 receptor, enabling antigen presentation, it is plausible that antigen-presentation takes place in the TME in relation to this finding. Currently we are investigating multiplex analyses on bladder tumors, urine and blood from tumor bearing animals to identify cytokines from the TME or systemically, that correlate to the formation of an adaptive immune response. For the liquid biopsy of the urine and serum we have performed a multiplex proteomic analysis using the proximity extension assay (PEA), an assay that greatly reduces the risk for cross-reactivity in the multiplex format. In a pilot analysis with PEA we noted that a single ortothopic instillation of CpG 1668 ODNs led to decreased TGFa and increased CCL3 in serum. This may reflect a change in the TME and a recruitment of cells into the tumor from the periphery, as a response to the pharmacological intervention. By this preliminary data we conclude that in the orthotopic MB49 tumor model, CCL21 appears to play a role in the induction of adaptive immune responses through the CCL21/CCR7 axis and that a single administration of locally acting TLR9 agonist into the bladder, can change the levels of systemic biomarkers. We will now further elucidate the early and late immunological events that lead to an effective immune response in this murine bladder cancer model. Citation Format: Iliana Kerzeli, Andrea Kurtinovic, Aikaterini Nasi, Ivan Stepanek, Martin Lord, Sara M. Mangsbo. Early immunological events in the periphery and the TME following a local immunostimulating instillation into the bladder in the MB49 orthotopic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 501.