No abstract

Fibroblasts are common cell types in cancer stroma and lay down collagen required for survival and growth of cancer cells. Although some cancer therapy strategies target tumor fibroblasts, their origin remains controversial. Multiple publications suggest circulating mesenchymal precursors as a source of tumor-associated fibroblasts. However, we show by three independent approaches that tumor fibroblasts derive primarily from local, sessile precursors. First, transplantable tumors developing in a mouse expressing green fluorescent reporter protein (EGFP) under control of the type I collagen (Col-I) promoter (COL-EGFP) had green stroma, whereas we could not find COL-EGFP+ cells in tumors developing in the parabiotic partner lacking the fluorescent reporter. Lack of incorporation of COL-EGFP+ cells from the circulation into tumors was confirmed in parabiotic pairs of COL-EGFP mice and transgenic mice developing autochthonous intestinal adenomas. Second, transplantable tumors developing in chimeric mice reconstituted with bone marrow cells from COL-EGFP mice very rarely showed stromal fibroblasts expressing EGFP. Finally, cancer cells injected under full-thickness COL-EGFP skin grafts transplanted in nonreporter mice developed into tumors containing green stromal cells. Using multicolor in vivo confocal microscopy, we found that Col-I–expressing fibroblasts constituted approximately one-third of the stromal mass and formed a continuous sheet wrapping the tumor vessels. In summary, tumors form their fibroblastic stroma predominantly from precursors present in the local tumor microenvironment, whereas the contribution of bone marrow-derived circulating precursors is rare.

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN-γ results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF-α and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-α, which was independent of NF-κB and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.

Immunogenic tumors grow progressively even when heavily infiltrated by CD8+ T cells. We investigated how to rescue CD8+ T cell function in long-established immunogenic melanomas that contained a high percentage of endogenous PD-1+ tumor-specific CD8+ T cells that were dysfunctional. Treatment with αPD-L1 and αCTLA-4 blocking antibodies did not prevent tumors from progressing rapidly. We then tested exogenous tumor-specific antigen delivery into tumors using Salmonella Typhimurium A1-R to increase antigen levels and generate a proinflammatory tumor microenvironment. Antigen-producing A1-R rescued the endogenous tumor-specific CD8+ T cell response: proliferation was induced in the lymphoid organs and effector function was recovered in the tumor. Treatment with antigen-producing A1-R led to improved mouse survival and resulted in 32% rejection of long-established immunogenic melanomas. Following treatment with antigen-producing A1-R, the majority of tumor-specific CD8+ T cells still expressed a high level of PD-1 in the tumor. Combining antigen-producing A1-R with αPD-L1 blocking antibody enhanced the expansion of tumor-specific CD8+ T cells and resulted in 80% tumor rejection. Collectively, these data demonstrate a powerful new therapeutic approach to rescue dysfunctional endogenous tumor-specific CD8+ T cells and eradicate advanced immunogenic tumors.

Purpose Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic non-synonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T cell receptor (TCR) that is specific for a single AAS. Experimental Design By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy. Results When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intra-tumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar. Conclusions Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape.

Cancer immunotherapy has been revolutionised by drugs that enhance the ability of the immune system to detect and fight tumors. Immune checkpoint therapies that target the programmed death-1 receptor (PD-1), or its ligand (PD-L1) have shown unprecedented rates of durable clinical responses in patients with various cancer types. However, there is still a large fraction of patients that do not respond to checkpoint inhibitors, and the challenge remains to find cellular and molecular cues that could predict which patients would benefit from these therapies. Using a series of qualitative and quantitative methods we show here that PBMCs and platelets from smokers and patients with head and neck squamous cell carcinoma (HNSCC) or lung cancer express and up-regulate PD-L1 independently of tumor stage. Furthermore, treatment with Atezolizumab, a fully humanised monoclonal antibody against PD-L1, in 4 patients with lung cancer caused a decrease in PD-L1 expression in platelets, which was restored over 20 days. Altogether, our findings reveal the expression of the main therapeutic target in current checkpoint therapies in human platelets and highlight their potential as biomarkers to predict successful therapeutic outcomes.

Purpose: Recurrent tumors (RT) of head and neck squamous cell carcinoma (HNSCC) occur in up to 60%, with poor therapeutic response and detrimental prognosis. We hypothesized that HNSCC RTs successfully evade antitumor immune response and aimed to reveal tumor immune microenvironment (TIME) changes of primary tumors (PT) and corresponding RTs. Experimental Design: Tumor-infiltrating leukocytes (TIL) of 300 PTs and 108 RTs from two large independent and clinically well-characterized HNSCC cohorts [discovery cohort (DC), validation cohort (VD)] were compared by IHC. mRNA expression analysis of 730 immune-related genes was performed for 18 PTs and RTs after adjuvant chemoradiotherapy (CRT). The effect of chemotherapy and radiation resistance was assessed with an in vitro spheroid/immunocyte coculture model. Results: TIME analysis revealed overall decrease of TILs with significant loss of CD8+ T cells (DC P = 0.045/VC P < 0.0001) and B lymphocytes (DC P = 0.036/VC P < 0.0001) in RTs compared with PTs in both cohorts. Decrease predominantly occurred in RTs after CRT. Gene expression analysis confirmed loss of TILs (P = 0.0004) and B lymphocytes (P < 0.0001) and showed relative increase of neutrophils (P = 0.018), macrophages (P < 0.0001), dendritic cells (P = 0.0002), and mast cells (P = 0.0057) as well as lower overall expression of immune-related genes (P = 0.018) in RTs after CRT. Genes involved in B-lymphocyte functions and number of tertiary lymphoid structures showed the strongest decrease. SPP1 and MAPK1 were upregulated in vivo and in vitro, indicating their potential suitability as therapeutic targets in CRT resistance. Conclusions: HNSCC RTs have an immunosuppressive TIME, which is particularly apparent after adjuvant CRT and might substantially contribute to poor therapeutic response and prognosis.

Intravital microscopy (IVM) offers the opportunity to visualize static and dynamic changes of tissue on a cellular level. It is a valuable tool in research and may considerably improve clinical diagnosis. In contrast to confocal and non-linear microscopy, optical coherence tomography (OCT) with microscopic resolution (mOCT) provides intrinsically cross-sectional imaging. Changing focus position is not needed, which simplifies especially endoscopic imaging. For in-vivo imaging, here we are presenting endo-microscopic OCT (emOCT). A graded-index-lens (GRIN) based 2.75 mm outer diameter rigid endoscope is providing 1.5 - 2 µm nearly isotropic resolution over an extended field of depth. Spherical and chromatic aberrations are used to elongate the focus length. Simulation of the OCT image formation, suggests a better overall image quality in this range compared to a focused Gaussian beam. Total imaging depth at a reduced sensitivity and lateral resolution is more than 200 µm. Using a frame rate of 80 Hz cross-sectional images of concha nasalis were demonstrated in humans, which could resolve cilial motion, cellular structures of the epithelium, vessels and blood cells. Mucus transport velocity was successfully determined. The endoscope may be used for diagnosis and treatment control of different lung diseases like cystic fibrosis or primary ciliary dyskinesia, which manifest already at the nasal mucosa.