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Conventional type 1 dendritic cells (cDC1s) are critical for innate sensing of cancer, yet they are scarce in the tumor microenvironment (TME). Here, we present a protocol to identify and isolate cDC1 subsets from murine implantable tumors for subsequent transcriptomic profiling using a flow sorting-based strategy. We describe steps for cell culture of mouse tumors, tumoral growth, dissociation and isolation of tumoral cells, extracellular staining, and cell sorting. We then detail procedures for RNA isolation, mRNA library preparation, and sequencing. For complete details on the use and execution of this protocol, please refer to Papadas et al.1.

Advances in immunotherapy in the last decade have revolutionized treatment paradigms across multiple cancer diagnoses. However, only a minority of patients derive durable benefit and progress with traditional approaches, such as cancer vaccines, remains unsatisfactory. A key to overcoming these barriers resides with a deeper understanding of tumor antigen presentation and the complex and dynamic heterogeneity of tumor-infiltrating antigen-presenting cells (APCs). Reminiscent of the 'second touch' hypothesis proposed by Klaus Ley for CD4+ T cell differentiation, the acquisition of full effector potential by lymph node- primed CD8+ T cells requires a second round of co-stimulation at the site where the antigen originated, i.e. the tumor bed. The tumor stroma holds a prime role in this process by hosting specialized APC niches, apparently distinct from tertiary lymphoid structures, that support second antigenic touch encounters and CD8+ T cell effector proliferation and differentiation. We propose that APC within second-touch niches become licensed for co-stimulation through stromal-derived instructive signals emulating embryonic or wound-healing provisional matrix remodeling. These immunostimulatory roles of stroma contrast with its widely accepted view as a physical and functional 'immune barrier'. Stromal control of antigen presentation makes evolutionary sense as the host stroma-tumor interface constitutes the prime line of homeostatic 'defense' against the emerging tumor. In this review, we outline how stroma-derived signals and cells regulate tumor antigen presentation and T-cell effector differentiation in the tumor bed. The re-definition of tumor stroma as immune rheostat rather than as inflexible immune barrier harbors significant untapped therapeutic opportunity.

Advances in immunotherapy in the last decade have revolutionized treatment paradigms across multiple cancer diagnoses. However, only a minority of patients derive durable benefit and progress with traditional approaches, such as cancer vaccines, remains unsatisfactory. A key to overcoming these barriers resides with a deeper understanding of tumor antigen presentation and the complex and dynamic heterogeneity of tumor-infiltrating antigen-presenting cells (APCs). Reminiscent of the 'second touch' hypothesis proposed by Klaus Ley for CD4+ T cell differentiation, the acquisition of full effector potential by lymph node- primed CD8+ T cells requires a second round of co-stimulation at the site where the antigen originated, i.e. the tumor bed. The tumor stroma holds a prime role in this process by hosting specialized APC niches, apparently distinct from tertiary lymphoid structures, that support second antigenic touch encounters and CD8+ T cell effector proliferation and differentiation. We propose that APC within second-touch niches become licensed for co-stimulation through stromal-derived instructive signals emulating embryonic or wound-healing provisional matrix remodeling. These immunostimulatory roles of stroma contrast with its widely accepted view as a physical and functional 'immune barrier'. Stromal control of antigen presentation makes evolutionary sense as the host stroma-tumor interface constitutes the prime line of homeostatic 'defense' against the emerging tumor. In this review, we outline how stroma-derived signals and cells regulate tumor antigen presentation and T-cell effector differentiation in the tumor bed. The re-definition of tumor stroma as immune rheostat rather than as inflexible immune barrier harbors significant untapped therapeutic opportunity., (© 2023 The Author(s).)

Cancer immunoediting progresses through elimination, equilibrium, and escape. Each of these phases is characterized by breaching, remodeling, and rebuilding tissue planes and structural barriers that engage extracellular matrix (ECM) components, in particular matrix proteoglycans. Some of the signals emanating from matrix proteoglycan remodeling are readily co-opted by the growing tumor to sustain an environment of tumor-promoting and immune-suppressive inflammation. Yet other matrix-derived cues can be viewed as part of a homeostatic response by the host, aiming to eliminate the tumor and restore tissue integrity. These latter signals may be harnessed for therapeutic purposes to tip the polarity of the tumor immune milieu toward anticancer immunity. In this review, we attempt to showcase the importance and complexity of matrix proteoglycan signaling in both cancer-restraining and cancer-promoting inflammation. We propose that the era of matrix diagnostics and therapeutics for cancer is fast approaching the clinic.