SUMMARY Complement is a critical component of humoral immunity implicated in cancer development, however its biological contributions to tumorigenesis remain poorly understood. Using the K14-HPV16 transgenic mouse model of squamous carcinogenesis, we report that urokinase (uPA)+ macrophages regulate C3-independent release of C5a during premalignant progression that in turn regulates protumorigenic properties of C5aR1+ mast cells and macrophages, including suppression of CD8+ T cell cytotoxicity. Therapeutic inhibition of C5aR1 via the peptide antagonist PMX-53 improved efficacy of paclitaxel chemotherapy associated with increased presence and cytotoxic properties of CXCR3+ effector memory CD8+ T cells in carcinomas, dependent on both macrophage transcriptional programming and IFNγ. Together, these data identify C5aR1-dependent signaling as an important immunomodulatory program in neoplastic tissue tractable for combinatorial cancer immunotherapy.

Immunotherapies targeting aspects of T cell functionality are efficacious in many solid tumors, but pancreatic ductal adenocarcinoma (PDAC) remains refractory to these treatments. Deeper understanding of the PDAC immune ecosystem is needed to identify additional therapeutic targets and predictive biomarkers for therapeutic response and resistance monitoring. To address these needs, we quantitatively evaluated leukocyte contexture in 135 human PDACs at single-cell resolution by profiling density and spatial distribution of myeloid and lymphoid cells within histopathologically defined regions of surgical resections from treatment-naive and presurgically (neoadjuvant)–treated patients and biopsy specimens from metastatic PDAC. Resultant data establish an immune atlas of PDAC heterogeneity, identify leukocyte features correlating with clinical outcomes, and, through an in silico study, provide guidance for use of PDAC tissue microarrays to optimally measure intratumoral immune heterogeneity. Atlas data have direct applicability as a reference for evaluating immune responses to investigational neoadjuvant PDAC therapeutics where pretherapy baseline specimens are not available. Significance: We provide a phenotypic and spatial immune atlas of human PDAC identifying leukocyte composition at steady state and following standard neoadjuvant therapies. These data have broad utility as a resource that can inform on leukocyte responses to emerging therapies where baseline tissues were not acquired. This article is highlighted in the In This Issue feature, p. 1861

Cancer-associated fibroblast (CAF) heterogeneity is increasingly appreciated, but the origins and functions of distinct CAF subtypes remain poorly understood. The abundant and transcriptionally diverse CAF population in pancreatic ductal adenocarcinoma (PDAC) is thought to arise from a common cell of origin, pancreatic stellate cells (PSCs), with diversification resulting from cytokine and growth factor gradients within the tumor microenvironment. Here we analyzed the differentiation and function of PSCs during tumor progression in vivo. Contrary to expectations, we found that PSCs give rise to a numerically minor subset of PDAC CAFs. Targeted ablation of PSC-derived CAFs within their host tissue revealed non-redundant functions for this defined CAF population in shaping the PDAC microenvironment, including production of specific extracellular matrix components and tissue stiffness regulation. Together, these findings link stromal evolution from distinct cells of origin to transcriptional heterogeneity among PDAC CAFs, and demonstrate unique functions for CAFs of a defined cellular origin.Statement of significance: By tracking and ablating a specific CAF population, we find that a numerically minor CAF subtype from a defined cell of origin plays unique roles in establishing the pancreatic tumor microenvironment. Together with prior studies, this work suggests that mesenchymal lineage heterogeneity as well as signaling gradients diversify PDAC CAFs.Research.

BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissues for RNA-seq have advantages over fresh frozen tissue including abundance and availability, connection to rich clinical data, and association with patient outcomes. However, FFPE-derived RNA is highly degraded and chemically modified, which impacts its utility as a faithful source for biological inquiry.MethodsTrue archival FFPE breast cancer cases (n = 58), stored at room temperature for 2–23 years, were utilized to identify key steps in tissue selection, RNA isolation, and library choice. Gene expression fidelity was evaluated by comparing FFPE data to public data obtained from fresh tissues, and by employing single-gene, gene set and transcription network-based regulon analyses.ResultsWe report a single 10 μm section of breast tissue yields sufficient RNA for RNA-seq, and a relationship between RNA quality and block age that was not linear. We find single-gene analysis is limiting with FFPE tissues, while targeted gene set approaches effectively distinguish ER+ from ER- breast cancers. Novel utilization of regulon analysis identified the transcription factor KDM4B to associate with ER+ disease, with KDM4B regulon activity and gene expression having prognostic significance in an independent cohort of ER+ cases.ConclusionOur results, which outline a robust FFPE-RNA-seq pipeline for broad use, support utilizing FFPE tissues to address key questions in the breast cancer field, including the delineation between indolent and life-threatening disease, biological stratification and molecular mechanisms of treatment resistance.

Cancers induced by human papillomaviruses (HPV) should be responsive to immunotherapy by virtue of expressing the immunogenic oncoproteins E6/E7. However, advanced forms of cervical cancer, driven by HPV, are poorly responsive to immune response–enhancing treatments involving therapeutic vaccination against these viral neoantigens. Leveraging a transgenic mouse model of HPV-derived cancers, K14HPV16/H2b, we demonstrated that a potent nanoparticle-based E7 vaccine, but not a conventional “liquid” vaccine, induced E7 tumor antigen–specific CD8+ T cells in cervical tumor–bearing mice. Vaccination alone or in combination with anti-PD-1/anti-CTLA4 did not elicit tumor regression nor increase CD8+ T cells in the tumor microenvironment (TME), suggesting the presence of immune-suppressive barriers. Patients with cervical cancer have poor dendritic cell functions, have weak cytotoxic lymphocyte responses, and demonstrate an accumulation of myeloid cells in the periphery. Here, we illustrated that myeloid cells in K14HPV16/H2b mice possess potent immunosuppressive activity toward antigen-presenting cells and CD8+ T cells, dampening antitumor immunity. These immune-inhibitory effects inhibited synergistic effects of combining our oncoprotein vaccine with immune checkpoint–blocking antibodies. Our data highlighted a link between HPV-induced cancers, systemic amplification of myeloid cells, and the detrimental effects of myeloid cells on CD8+ T-cell activation and recruitment into the TME. These results established immunosuppressive myeloid cells in lymphoid organs as an HPV+ cancer–induced means of circumventing tumor immunity that will require targeted abrogation to enable the induction of efficacious antitumor immune responses.