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Immune checkpoint inhibitors targeting the interaction between programmed cell death-1 (PD-1) and its ligand PD-L1 induce tumor regression in a subset of non-small cell lung cancer patients. However, clinical response rates are less than 25%. Evaluation of combinations of immunotherapy with existing therapies requires appropriate pre-clinical animal models. In this study, murine lung cancer cells (CMT167 and LLC) were implanted either orthotopically in the lung or subcutaneously in syngeneic mice, and response to anti-PD-1/PD-L1 therapy was determined. Anti-PD-1/PD-L1 therapy inhibited CMT167 orthotopic lung tumors by 95%The same treatments inhibited CMT167 subcutaneous tumors by only 30%, and LLC orthotopic lung tumors by 35%. CMT167 subcutaneous tumors had more Foxp3+ CD4+ T cells and fewer PD-1+ CD4+ T cells compared to CMT167 orthotopic tumors. Flow cytometric analysis also demonstrated increased abundance of PD-L1high cells in the tumor microenvironment in CMT167 tumor-bearing lungs compared to CMT167 subcutaneous tumors or LLC tumor-bearing lungs. Silencing PD-L1 expression in CMT167 cells resulted in smaller orthotopic tumors that remained sensitive to anti-PD-L1 therapy, whereas implantation of CMT167 cells into PD-L1− mice blocked orthotopic tumor growth, indicating a role for PD-L1 in both the cancer cell and the microenvironment. These findings indicate that the response of cancer cells to immunotherapy will be determined by both intrinsic properties of the cancer cells and specific interactions with the microenvironment. Experimental models that accurately recapitulate the lung tumor microenvironment are useful for evaluation of immunotherapeutic agents.

Rationale The vascular adventitia is a complex layer of the vessel wall consisting of vasa vasorum microvessels, nerves, fibroblasts, immune cells, and resident progenitor cells. Adventitial progenitors express the stem cell markers, Sca1 and CD34 (AdvSca1 cells), have the potential to differentiate in vitro into multiple lineages, and potentially contribute to intimal lesions in vivo. Objective While emerging data support the existence of AdvSca1 cells, the goal of this study was to determine their origin, degree of multipotency and/or heterogeneity, and contribution to vessel remodeling. Methods and Results Using two in vivo fate-mapping approaches combined with a smooth muscle cell (SMC) epigenetic lineage mark, we report that a subpopulation of AdvSca1 cells is generated in situ from differentiated SMCs. Our data establish that the vascular adventitia contains phenotypically distinct subpopulations of progenitor cells expressing SMC, myeloid, and hematopoietic progenitor-like properties and that differentiated SMCs are a source to varying degrees of each subpopulation. SMC-derived AdvSca1 cells exhibit a multipotent phenotype capable of differentiating in vivo into mature SMCs, resident macrophages, and endothelial-like cells. Following vascular injury, SMC-derived AdvSca1 cells expand in number and are major contributors to adventitial remodeling. Induction of the transcription factor Klf4 in differentiated SMCs is essential for SMC reprogramming in vivo while in vitro approaches demonstrate that Klf4 is essential for maintenance of the AdvSca1 progenitor phenotype. Conclusions We propose that generation of resident vascular progenitor cells from differentiated SMCs is a normal physiological process that contributes to the vascular stem cell pool and plays important roles in arterial homeostasis and disease.

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The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3 mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ/TNFα/IL10 CD4 and CD8 T cells. Immunodepletion of CD4 but not CD8 T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5-dependent pathway. This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. .