Ovarian cancer stem cells and macrophages reciprocally interact through the WNT pathway to promote pro-tumoral and malignant phenotypes in 3D engineered microenvironments

Abstract

Background Innate immune cells such as macrophages are abundantly present within malignant ascites, where they share the microenvironment with ovarian cancer stem cells (CSC). Methods To mimic this malignant ascites microenvironment, we created a hanging-drop hetero-spheroid model to bring CSCs and macrophages in close association. Within these hetero-spheroids, CD68 + macrophages (derived from U937 or peripheral blood monocytes) make up ~ 20% of the population, while the rest are ovarian cancer cells and ovarian cancer stem cells (derived from the high grade serous ovarian cancer cell line, OVCAR3). Results Our results indicate that CSCs drive the upregulation of M2 macrophage marker CD206 within hetero-spheroids, compared to bulk ovarian cancer cells, implying an inherently more immuno-suppressive program. Moreover, an increased maintenance of elevated aldehyde dehydrogenase (ALDH) activity is noted within hetero-spheroids that include pre-polarized CD206 + M2 macrophages, implying a reciprocal interaction that drives pro-tumoral activation as well as CSC self-renewal. Consistent with enriched CSCs, we also observe increased levels of pro-tumoral IL-10 and IL-6 cytokines in the CSC/M2-macrophage hetero-spheroids. CSC/M2-macrophage hetero-spheroids are also less sensitive to the chemotherapeutic agent carboplatin and are subsequently more invasive in transwell assays. Using inhibitors of WNT secretion in both CSCs and macrophages, we found that CSC-derived WNT ligands drove CD206 + M2 macrophage activation, and that, conversely, macrophage-derived WNT ligands enriched ALDH + cells within the CSC compartment of hetero-spheroids. Upon examination of specific WNT ligand expression within the monocyte-derived macrophage system, we observed a significant elevation in gene expression for WNT5B . In CSCs co-cultured with macrophages within hetero-spheroids, increases in several WNT ligands were observed, and this increase was significantly inhibited when WNT5B was knocked down in macrophages. Conclusions Our data implies that macrophage- initiated WNT signaling could play a significant role in the maintenance of stemness, and the resulting phenotypes of chemoresistance and invasiveness. Our results indicate paracrine WNT activation during CSC/M2 macrophages interaction constitutes a positive feedback loop that likely contributes to the more aggressive phenotype, which makes the WNT pathway a potential target to reduce the CSC and M2 macrophage compartments in the tumor microenvironment. Electronic supplementary material The online version of this article (10.1186/s40425-019-0666-1) contains supplementary material, which is available to authorized users.

Carcinoma-Associated Mesenchymal Stem Cells Promote Chemoresistance in Ovarian Cancer Stem Cells via PDGF Signaling

“…Given the heterogeneity in the patient samples, the IC 50 values differed significantly between the heterospheroids generated with the OVCAR3 CSC and patient-derived CSC. These results are expected based on the previous characterization of patient-derived heterogeneity in the hanging drop spheroids [18][19][20][21]55].…”

“…To assess the impact of MSC on cancer cell stemness, we evaluated aldehyde dehydrogenase (ALDH) activity within heterospheroids as a marker of CSC, consistent with our prior work [28]. Previous work has established ovarian cancer cells that express CD133 highly and have elevated ALDH activity, as the ALDH+ CD133+ CSC, which are at the apex of the CSC hierarchy [8,9,11,[18][19][20][21]55,59]. These CSC and can self-renew and asymmetrically divide to create daughter progenies that are either ALDH+ or CD133+.…”

“…Chemoresistance and new tumor growth following primary therapy has been ascribed to ovarian cancer stem cells (CSC), through a complex interplay with other cells within the tumor microenvironment (TME) [1,[3][4][5][6][7]. Previous studies have indicated that CSC in ovarian cancer are identified with high CD133 expression and increased aldehyde dehydrogenases (ALDH) activity, and presence of these cells predicts prognosis for reduced progression-free survival and poor patient outcome [8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Additionally, elevated ALDH activity alone has been utilized in previous studies as a marker of the CSC progenies [8][9][10][11][12][13][14][15][16][17][18][19][20][21].…”

Targeting Tumor-Associated Macrophages in Anti-Cancer Therapies: Convincing the Traitors to Do the Right Thing

“…CSCs are a population of tumor cells, which share some features with stem cells, being able to initiate tumorigenesis thanks to their ability for continuous self-renewal and differentiation [ 37 ]. In this context, TAMs produce soluble factors (e.g., TGF-β, IL-6) that promote survival of CSCs [ 20 , 21 , 38 , 39 ]. Our group has recently found that also GPNMB produced by macrophages induces cancer stemness via CD44 binding and release of IL-33 [ 40 ].…”

Expansion of Circulating Tumor Cells from Patients with Locally Advanced Pancreatic Cancer Enable Patient Derived Xenografts and Functional Studies for Personalized Medicine

“…Cells were removed from culture surfaces by gentle trituration and collected in conical tubes. Pancreatic CTC spheroids were generated on 384 well hanging drop array plates, following extensively established protocols [37][38][39][40]. Briefly, cell suspensions were adjusted such that 20 µL contained 100 pancreatic CTCs.…”