The Cancer Cell Dissemination Machinery as an Immunosuppressive Niche: A New Obstacle Towards the Era of Cancer Immunotherapy

Abstract

Although cancer immunotherapy has resulted in unpreceded survival benefits to subsets of oncology patients, accumulating evidence from preclinical animal models suggests that the immunosuppressive tumor microenvironment remains a detrimental factor limiting benefit for many patient subgroups. Recent efforts on lymphocyte-mediated immunotherapies are primarily focused on eliminating cancer foci at primary and metastatic sites, but few studies have investigated the impact of these therapies on the highly complex process of cancer cell dissemination. The metastatic cascade involves the directional streaming of invasive/migratory tumor cells toward specialized blood vessel intravasation gateways, called TMEM doorways, to the peripheral circulation. Importantly, this process occurs under the auspices of a specialized tumor microenvironment, herewith referred to as "Dissemination Trajectory", which is supported by an ample array of tumor-associated macrophages (TAMs), skewed towards an M2-like polarization spectrum, and which is also vital for providing microenvironmental cues for cancer cell invasion, migration and stemness. Based on pre-existing evidence from preclinical animal models, this article outlines the hypothesis that dissemination trajectories do not only support the metastatic cascade, but also embody immunosuppressive niches, capable of providing transient and localized immunosubversion cues to the migratory/invasive cancer cell subpopulation while in the act of departing from a primary tumor. So long as these dissemination trajectories function as "immune deserts", the migratory tumor cell subpopulation remains efficient in evading immunological destruction and seeding metastatic sites, despite administration of cancer immunotherapy and/or other cytotoxic treatments. A deeper understanding of the molecular and cellular composition, as well as the signaling circuitries governing the function of these dissemination trajectories will further our overall understanding on TAM-mediated immunosuppression and will be paramount for the development of new therapeutic strategies for the advancement of optimal cancer chemotherapies, immunotherapies, and targeted therapies.

Keywords: cancer immunotherapy; T cells; endothelial anergy; lymphocyte exclusion; lymphocyte exhaustion; macrophages; metastasis; tumor microenvironment.

Figure 1

The “Dissemination Trajectory” Working Model of Metastatic Dissemination. Two major cellular prerequisites are necessary for cancer cell dissemination: a TMEM doorway and a highly invasive, highly migratory cancer cell subsets streaming toward TMEM doorways. TMEM doorways are composed of three cell types, a TIE2⁺ macrophage, an endothelial cell and a tumor cell forming an invadopod in the vasculature, and signaling conversation among these three cells results in localized vascular opening to facilitate transendothelial migration of the highly invasive, highly migratory cancer cell subset. The highly invasive and migratory cancer cell subsets participate in a reciprocal paracrine and juxtacrine signaling loop with intratumoral macrophages that do not express TIE2, resulting in the increased induction of the actin-regulatory protein MENA^INV. Eventually, these interactions result in the so called “streaming migration”, which is directed toward TMEM doorways, and MENA^INV-facilitated transendothelial migration and metastatic dissemination. TMEM doorways and their streaming MENA^INV+ cancer cell subsets are herewith referred to as “dissemination trajectories”. These specialized microenvironments are distinguishable from other tumor compartments with rapidly dividing tumor cells that do not share similar molecular pathways, here described as “proliferative compartments”. Four layers of immunosuppressive mechanisms dominate within the dissemination trajectories, that result in the development of immune deserts further facilitating the process of metastatic dissemination. These mechanisms postulate that: (a) the TMEM endothelium is anergic, thus not allowing for T cell diapedesis; (b) dissemination trajectories do not support cytokine/chemokine matching for allowing T cell chemotaxis; (c) dissemination trajectories have a unique metabolic landscape that is refractory for T cell chemotaxis and/or function; and finally (d) dissemination trajectories are characterized by the induction of immune checkpoint signaling, that promoted exhaustion of T cells. Overall, tumor-associated macrophages (TAMs) within these dissemination trajectories play the pivotal role in regulating all four layers of immunosuppression, although secondary mechanisms have also been identified.

Figure 2

Immunohistochemical indication of how different pharmacologic modifications of the immunosuppressive tumor microenvironment may affect T cell trafficking into tumors. (A–D) Immunohistochemistry for T cell specific marker CD3 in tumor sections from mouse mammary tumor virus – polyoma middle T antigen (MMTV-PyMT) mice, developing spontaneous breast carcinomas. The images are high power fields (x40), representative from a total of three mice in each experimental condition. Circles, CD3⁺ T cells infiltrating the tumor nests; Arrows, CD3⁺ T cells infiltrating the tumor stroma. Notice the significant changes in intratumoral versus stromal T cell infiltration upon different treatments that modify the immunosuppressive microenvironment.) In breast carcinoma, T cells are found in both tumor cell nests and the tumor stroma (A). Upon macrophage depletion with clodronate liposomes, most T cells can leave the stroma and penetrate the tumor cell nests (B). However, treatment with cytotoxic chemotherapy is known to induce lymphocyte infiltration and significantly larger number of T cells is found compared to the vehicle (C). Notably however, most of these T cells are restricted in the tumor stroma, as chemotherapy attracts immunosuppressive myeloid cells at the same time, resulting in lymphocyte exclusion (C). If such immunosuppressive myeloid cells are depleted through clodronate liposomes in chemotherapy-treated tumors, the increased influx of T cells is now relocated in the tumor nests (D). Immunohistochemistry was performed in archival tissue from experiments originally conducted in the manuscript by Karagiannis et al. (139), in which ethical approval for the use of the experimental mice was also obtained (139).

Figure 3

Proposed Mechanisms for the Induction and Maintenance of an Immunosuppressive Microenvironment within the Dissemination Trajectory. (A) Dissemination trajectories as beacons of endothelial anergy. Perivascular (TMEM doorway) macrophages secrete a number of proangiogenic factors (e.g. VEGFA) in the peri-TMEM area, which downregulate cell adhesion molecules in endothelial cells critical for lymphocyte diapedesis, thus resulting in “locally” anergic endothelium. (B) Dissemination trajectories as crossroads for T cell exclusion. Cytokine/cytokine receptor mismatching mechanisms within the dissemination trajectories result in the exclusion of T cells. For example, prometastatic macrophages suppress the expression CXCL9/10 within the dissemination trajectories, which function as the primary chemoattractants for T cells. Instead, dissemination trajectories are characterized by the expression of other cytokines/chemokines, like TGF-beta and CXCL12, which act as repellents for T cells. (C) Dissemination trajectories as primers for metabolic burdening of T cells. Highly migratory tumor cells within the dissemination trajectories tend to upregulate glucose transporters (e.g., GLUT1), which on one hand reduces the bioavailable energy resources (i.e., glucose), while on the other hand, may produce metabolites. This metabolic landscape is burdensome for immune cells, resulting in T cell exclusion and exhaustion. (D) Dissemination trajectories as checkpoints for T cell exhaustion. Chronic TCR signaling within the dissemination trajectory along with overexpression of inhibitory ligands (e.g., PDL1) by the prometastatic macrophages may result in T-regulatory (Treg) cell expansion and CD8+ T cell inactivation/exhaustion.