Cholangiocarcinoma stem-like subset shapes tumor-initiating niche by educating associated macrophages

Abstract

Background & Aims A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. Methods CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium. Results CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p = 0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n = 23) and correlated with a high level of serum cancer antigen 19.9 (n = 17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n = 12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n = 104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect. Conclusion CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease. Lay summary Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like ...

Molecular Mechanisms Driving Cholangiocarcinoma Invasiveness: An Overview

“…In CCA tissue, TAMs are predominantly localized at the tumor front 37 , and their number was shown to positively correlate with the presence of extrahepatic metastases 38 . Furthermore, incubation of CCA cells with conditioned medium from M2 macrophages increased cancer cell migration, while promoting EMT-like changes 38 .…”

Tumor reactive stroma in cholangiocarcinoma: The fuel behind cancer aggressiveness

“…They found that, in CCA tissues, the number of CD163 + M2 cells was, as expected, lower than the number of CD68 + cells, and that high infiltration of M2 macrophages, but not of total macrophages, was significantly associated with poor disease-free survival of patients. Of further interest, the density of M2 macrophages within CCA stroma also correlated with the presence of extrahepatic metastases[28], the tumor pathological grade[67], and the microvascular density[64]. Although these findings are based on the evaluation of different phenotypic markers, overall, they suggest that TAMs strongly influence CCA progression, with a major role played by M2 TAMs, thus confirming what observed in other cancer types.…”

“…In CCA tissues, M2 macrophages are definitely much more abundant than in the peritumoral areas, and TAM are mostly located at the leading edge of the tumor[67]. Consistently, immunohistochemical analyses in Opisthorchis viverrini -associated CCA in a hamster model revealed a progressive, dramatic increase in M2 macrophages through carcinogenesis[28].…”

“…In line with this concept, many tumor-promoting cytokines that are actually M1 cytokines, such as IL-6, are even produced by TAMs[61]. Recently, Raggi et al[67] revealed that the CCA stem-like compartment is actively involved in both the recruitment of circulating monocytes and their differentiation into TAMs, owing to the release of IL-13, IL-34 and osteoactivin. Of note, cancer stem cell (CSC)-associated TAMs display unique phenotypic and functional features, namely mixed expression of M1 and M2 markers ( e.g ., M1-related chemokines CXCL9 and CXCL10, and M2-related chemokines CCL17 and CCL18), increased adhesive and invasive abilities, in vitro , and enhanced tumor-promoting functions, in vivo .…”

“…Moreover, CCA patients with high numbers of MMP-9 + TAMs displayed significantly shorter survival times than those with low numbers, thus pointing out MMP-9 production as a key driver of CCA progression promoted by TAMs. Furthermore, a broad expression of other pivotal ECM remodeling-related genes, namely MMP-2 , ADAM10 , and ADAM17 was reported in CSC-associated TAMs[67]. …”