Protocol for transcutaneous tumor photolabeling to track immune cells in vivo using Kaede mice.

Preclinical tumor models have advanced our understanding of the tumor microenvironment. However, the temporal dynamics of cellular recruitment and retention within these models is poorly understood. Here, we present a protocol using transcutaneous labeling of the tumor compartment using subcutaneous and orthotopic tumors. We describe the process of cell line implantation and photoconversion of tumors to differentiate newly recruited cells from those retained within tumors. Photoconversion enables tracking of both immune cell recruitment to tumors and egress to the lymphatics. For complete details on the use and execution of this protocol, please refer to Li et al.1 and Molostvov et al.2.

Rapid functional impairment of natural killer cells following tumor entry limits anti-tumor immunity.

Immune cell dysfunction within the tumor microenvironment (TME) undermines the control of cancer progression. Established tumors contain phenotypically distinct, tumor-specific natural killer (NK) cells; however, the temporal dynamics, mechanistic underpinning and functional significance of the NK cell compartment remains incompletely understood. Here, we use photo-labeling, combined with longitudinal transcriptomic and cellular analyses, to interrogate the fate of intratumoral NK cells. We reveal that NK cells rapidly lose effector functions and adopt a distinct phenotypic state with features associated with tissue residency. NK cell depletion from established tumors did not alter tumor growth, indicating that intratumoral NK cells cease to actively contribute to anti-tumor responses. IL-15 administration prevented loss of function and improved tumor control, generating intratumoral NK cells with both tissue-residency characteristics and enhanced effector function. Collectively, our data reveals the fate of NK cells after recruitment into tumors and provides insight into how their function may be revived.

Tumour-retained activated CCR7+ dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity.

Tumour dendritic cells (DCs) internalise antigen and upregulate CCR7, which directs their migration to tumour-draining lymph nodes (dLN). CCR7 expression is coupled to an activation programme enriched in regulatory molecule expression, including PD-L1. However, the spatio-temporal dynamics of CCR7+ DCs in anti-tumour immune responses remain unclear. Here, we use photoconvertible mice to precisely track DC migration. We report that CCR7+ DCs are the dominant DC population that migrate to the dLN, but a subset remains tumour-resident despite CCR7 expression. These tumour-retained CCR7+ DCs are phenotypically and transcriptionally distinct from their dLN counterparts and heterogeneous. Moreover, they progressively downregulate the expression of antigen presentation and pro-inflammatory transcripts with more prolonged tumour dwell-time. Tumour-residing CCR7+ DCs co-localise with PD-1+CD8+ T cells in human and murine solid tumours, and following anti-PD-L1 treatment, upregulate stimulatory molecules including OX40L, thereby augmenting anti-tumour cytolytic activity. Altogether, these data uncover previously unappreciated heterogeneity in CCR7+ DCs that may underpin a variable capacity to support intratumoural cytotoxic T cells.

Migration of stem-like CD8 T cells between tissue microenvironments underpins successful anti-tumour immune responses.

The clinical success of immune checkpoint blockade in some patients has transformed treatment approaches in cancer and offers the hope of durable curative responses. Building from studies of chronic infection, the composition of tumour infiltrating lymphocytes and in particular, the spectrum of exhausted CD8 T cells has now been characterized in detail, profiling the phenotype, function, transcriptional regulation and even the epigenetic changes. However, what remains less clear is how intratumoural immune cells interface with populations in the periphery, both in terms of sustaining the response in cancer, but also in establishing systemic memory responses that can provide long-term protection. Here we will succinctly review the current understanding of the anti-tumour response, consider the tissue microenvironments that support key cellular subsets and the extent to which cellular migration between these sites impacts the response.