Background Tumor progression is accompanied by dramatic remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness. The altered ECM of the tumor supports cancer growth and metastasis, but it is unknown if this effect involves modulation of T cell activity. To investigate if a high-density tumor-specific ECM could influence the ability of T cells to kill cancer cells, we here studied how T cells respond to 3D culture in different collagen densities. Methods T cells cultured in 3D conditions surrounded by a high or low collagen density were imaged using confocal fluorescent microscopy. The effects of the different collagen densities on T cell proliferation, survival, and differentiation were examined using flow cytometry. Cancer cell proliferation in similar 3D conditions was also measured. Triple-negative breast cancer specimens were analyzed for the number of infiltrating CD8+ T cells and for the collagen density. Whole-transcriptome analyses were applied to investigate in detail the effects of collagen density on T cells. Computational analyses were used to identify transcription factors involved in the collagen density-induced gene regulation. Observed changes were confirmed by qRT-PCR analysis. Results T cell proliferation was significantly reduced in a high-density matrix compared to a low-density matrix and prolonged culture in a high-density matrix led to a higher ratio of CD4+ to CD8+ T cells. The proliferation of cancer cells was unaffected by the surrounding collagen-density. Consistently, we observed a reduction in the number of infiltrating CD8+ T-cells in mammary tumors with high collagen-density indicating that collagen-density has a role in regulating T cell abundance in human breast cancer. Whole-transcriptome analysis of 3D-cultured T cells revealed that a high-density matrix induces downregulation of cytotoxic activity markers and upregulation of regulatory T cell markers. These transcriptional changes were predicted to involve autocrine TGF-β signaling and they were accompanied by an impaired ability of tumor-infiltrating T cells to kill autologous cancer cells. Conclusions Our study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process.

During tumor growth the extracellular matrix (ECM) undergoes dramatic remodeling. The normal ECM is degraded and substituted with a tumor-specific ECM, which is often of higher collagen density and increased stiffness. The structure and collagen density of the tumor-specific ECM has been associated with poor prognosis in several types of cancer. However, the reason for this association is still largely unknown. Collagen can promote cancer cell growth and migration, but recent studies have shown that collagens can also affect the function and phenotype of various types of tumor-infiltrating immune cells such as tumor-associated macrophages (TAMs) and T cells. This suggests that tumor-associated collagen could have important immune modulatory functions within the tumor microenvironment, affecting cancer progression as well as the efficacy of cancer immunotherapy. The effects of tumor-associated collagen on immune cells could help explain why a high collagen density in tumors is often correlated with a poor prognosis. Knowledge about immune modulatory functions of collagen could potentially identify targets for improving current cancer therapies or for development of new treatments. In this review, the current knowledge about the ability of collagen to influence T cell activity will be summarized. This includes direct interactions with T cells as well as induction of immune suppressive activity in other immune cells such as macrophages. Additionally, the potential effects of collagen on the efficacy of cancer immunotherapy will be discussed.

The development of immune checkpoint inhibitors (ICI) marks an important breakthrough of cancer therapies in the past years. However, only a limited fraction of patients benefit from such treatments, prompting the search for immune modulating agents that can improve the therapeutic efficacy. The nonselective beta blocker, propranolol, which for decades has been prescribed for the treatment of cardiovascular conditions, has recently been used successfully to treat metastatic angiosarcoma. These results have led to an orphan drug designation by the European Medicines Agency for the treatment of soft tissue sarcomas. The anti-tumor effects of propranolol are suggested to involve the reduction of cancer cell proliferation as well as angiogenesis. Here, we show that oral administration of propranolol delays tumor progression of MCA205 fibrosarcoma model and MC38 colon cancer model and increases the survival rate of tumor bearing mice. Propranolol works by reducing tumor angiogenesis and facilitating an anti-tumoral microenvironment with increased T cell infiltration and reduced infiltration of myeloid-derived suppressor cells (MDSCs). Using T cell deficient mice, we demonstrate that the full anti-tumor effect of propranolol requires the presence of T cells. Flow cytometry-based analysis and RNA sequencing of FACS-sorted cells show that propranolol treatment leads to an upregulation of PD-L1 on tumor associated macrophages (TAMs) and changes in their chemokine expression profile. Lastly, we observe that the co-administration of propranolol significantly enhances the efficacy of anti-CTLA4 therapy. Our results identify propranolol as an immune modulating agent, which can improve immune checkpoint inhibitor therapies in soft tissue sarcoma patients and potentially in other cancers.

Physiologic turnover of interstitial collagen is mediated by a sequential pathway in which collagen is fragmented by pericellular collagenases, endocytosed by collagen receptors, and routed to lysosomes for degradation by cathepsins. Here, we use intravital microscopy to investigate if malignant tumors, which are characterized by high rates of extracellular matrix turnover, utilize a similar collagen degradation pathway. Tumors of epithelial, mesenchymal, or neural crest origin all display vigorous endocytic collagen degradation. The cells engaged in this process are identified as tumor-associated macrophage (TAM)-like cells that degrade collagen in a mannose receptor-dependent manner. Accordingly, mannose-receptor-deficient mice display increased intratumoral collagen. Whole-transcriptome profiling uncovers a distinct extracellular matrix-catabolic signature of these collagen-degrading TAMs. Lineage-ablation studies reveal that collagen-degrading TAMs originate from circulating CCR2+ monocytes. This study identifies a function of TAMs in altering the tumor microenvironment through endocytic collagen turnover and establishes macrophages as centrally engaged in tumor-associated collagen degradation.

The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis of large collagen fragments. First, we show that collagen that has been pre-cleaved by a mammalian collagenase is taken up much more efficiently than intact, native collagen by uPARAP/Endo180-positive cells. Second, we demonstrate that this preference is governed by the acquisition of a gelatin-like structure by the collagen, occurring upon collagenase-mediated cleavage under native conditions. Third, we demonstrate that the growth of uPARAP/Endo180-deficient fibroblasts on a native collagen matrix leads to substantial extracellular accumulation of well defined collagen fragments, whereas, wild-type fibroblasts possess the ability to direct an organized and complete degradation sequence comprising both the initial cleavage, the endocytic uptake, and the intracellular breakdown of collagen.