Cell lines were purchased from ATCC (Manassas, VA). Peripheral blood mononuclear cells (PBMCs) were purified from buffy coats from healthy donors through Ficoll-Paque gradient centrifugation. Cytokines were purchased from Peprotech Inc., and all other tissue culture supplements from Life Technologies, and chemicals from Sigma Aldrich (St. Louis, MO) unless otherwise specified. Compounds Ruxolitinib and sc144 were a generous gift from the laboratory of Dr. Karen McLean. Viral vectors were purchased from Sigma Aldrich and packaged at the University of Michigan Viral Vector core.

U937 cells were cultured in suspension in RPMI supplemented with 10% heat-inactivated fetal calf serum (Atlanta Biologics) and 1x antibiotics/antimycotics. Cells were harvested, and suspended at 2500cells/ml and treated with 5 ng/ml phorbol myristate acetate (PMA). 20 μl of this suspension was plated onto each well of a hanging drop array plate, to allow monocytes to differentiate into macrophages in suspension culture. For PBMCs, cells were plated onto tissue culture dishes, and the non-adherent cell fraction was discarded following 24 h of attachment. PBMCs were then detached from the plate, and plated onto hanging drop arrays at 500 cells/drop. At the end of 24 h, each well was left untreated to derive M0 resting macrophages, or treated with 20 ng/ml recombinant human M-CSF and 20 ng/ml IL-4 to derive activated M2-like macrophages for the next 48 h. For brevity, figure captions refer to IL-4/MCSF activated macrophages as M2, to indicate the M2-like, alternatively activated phenotype. Macrophages were harvested from hanging drops and assessed for differentiation and polarization using flow analysis, described below. Harvested macrophage aggregates were also subjected to subsequent qPCR analysis, or used to manufacture hetero-spheroids.

Ovarian CSCs were isolated from the serous ovarian cancer cell lines OVCAR3 (used under passage 35) as described previously [13]. Briefly, cells were harvested and incubated with ALDEFLUOR reagent, and CD133 antibody, and sorted using flow cytometry for cells positive for elevated ALDH and CD133 positivity. Appropriate DEAB and isotype controls were used for both assays, to determine gate settings as described previously. CSCs were freshly sorted and used to make hetero-spheroids < 24 h after flow sorting.

Spheroids were generated on a hanging drop array plate from CSCs and macrophages adapting protocols described previously [13–15]. For mono-spheroids, 100 CSCs or the unsorted bulk of OVCAR3 cells were seeded per hanging drop and allowed to form spheroids. Macrophages were harvested from hanging drops following the differentiation protocol described in Section 2.2. Macrophages and CSCs were combined and plated onto hanging drop arrays such that each drop contained 100 CSCs and 100 M0/M2 macrophages. Mono-spheroids contained 100 CSCs or bulk unsorted OVCAR3 cells. Following 4–5 days in hanging drop array culture, spheroid formation was tracked using live cell microscopy and routinely fed to maintain a ~ 20 μl drop volume. Spheroids were used for subsequent flow analysis, qPCR or lysed to obtain protein for immunoblots. In some instances, hetero-spheroids were manufactured from CSCs stably expressing GFP, and fluorescent activated cell sorting was utilized to separate the GFP⁺ CSC compartment from hetero-spheroids for further analysis.

Flow cytometry analysis was performed following protocols established in our lab previously [13]. CD68-APC (Miltenyi Biotech, Germany) antibody was used, with its associated APC-isotype control, to identify CD68⁺ macrophages, in differentiated U937 or PBMC monocytes, and hetero-spheroids. For CD68 flow analysis, samples were fixed in methanol at − 20 °C, for 1 h, followed by a PBS wash to remove methanol, re-suspension in FACS Buffer (PBS + 2% FBS) before antibody incubation.

Two kinds of flow cytometry based experiments were carried out to characterize hetero-spheroids: i) macrophage polarization was assessed by CD68⁺ and CD206⁺ using flow cytometry; ii) Stemness was assessed by ALDEFLUOR assay for observation of elevated ALDH activity using flow cytometry, using protocols established previously [13]. Briefly, hetero-spheroids were harvested in FACS buffer, and triturated to single-cell suspensions. Appropriate isotype controls were used for conjugated antibodies, to set gates to observe CD68 and CD206. For ALDH, a molar excess of the DEAB inhibitor was used to determine positive gates per the manufacturer’s protocol, to identify elevated ALDH activity. ALDH activity was assessed following 48-h treatment with the JAK1/2 inhibitor, Ruxolitinib, or the GP130 inhibitor, sc144, or the human anti IL-6 antibody, Tocilizumab (Actemra, Genentech). Flow cytometry was performed on the Attune acoustic focusing flow cytometer (Applied Biosystems). Flow sorting was performed on the Astrios (Beckman Coulter).

RNA was extracted from harvested macrophages or hetero-spheroids using the RNeasy extraction kit (Qiagen). Extracted RNA was assessed for concentration and purity using a Nanodrop 2000 (Thermo Fisher Scientific) spectrophotometer. RNA was transcribed to cDNA using the High-fidelity cDNA Transcription kit (Life Technologies), and qPCR was carried out in the 96-well format using the 7900HT platform (Applied Biosystems). For macrophage polarization, CD163 and CD206 were assessed. IL-10 was additionally assessed in hetero-spheroids. Lastly, WNT ligands were also assessed in hetero-spheroids. Gene expression differences were quantified using the 2ΔΔC_T method, using GAPDH as the housekeeping control, and reported as fold change compared to a control sample. For macrophages, controls were undifferentiated monocytes. For hetero-spheroids, control samples were bulk OVCAR3 spheroids. qPCR experiments were run in triplicates, with 2–3 independent samples. A list of primers used in the qPCR experiments is provided in Additional file 1: Table S1.

For ELISA assays, media was harvested from 50 spheroids (macrophages, OVCAR3, CSC, CSC/M2 or CSC/sh-WNT5B M2). ELISA assays were performed on the Duoset ELISA system (R&D Biosystems, Minneapolis MN) following the manufacturer’s protocol, modified to include an overnight sample incubation. Cytokines analyzed included IL-10 and IL-6. Standard curves were generated for each cytokine, and analyte concentration was assessed using a four parametric ELISA curve, to determine the amount of IL-10 or IL-6 released. ELISA assays and data analysis were performed at the Immunological Monitoring Core at the Rogel Cancer Center, University of Michigan.

For chemoresistance, hetero-spheroids were treated with carboplatin, to a final concentration of 300 μM, within 20 μl drops, for 48 h. At the end of 48 h, the MTS reagent (Abcam) was added to drops at a 1/10 dilution, and allowed to incubate at 37 °C for 2.5 h. At the end of the incubation period, absorbance was read on the hetero-spheroids at 590 nm, according to manufacturer’s protocols. Untreated hetero-spheroids were used as controls, to normalize the absorbance to identify the effect of drug treatment on cellular viability. Results were quantified as normalized cell viability, based on untreated controls. Experiments were repeated with 3–5 biological replicates for statistical analysis.

In order to quantify invasiveness of hetero-spheroids, 8 μm transwell inserts were placed in each well of a 24 well plate. 10 CSC mono-spheroids or CSC/M2, CSC/scramble M2, CSC/sh-WNT5B M2 hetero-spheroids were harvested at Day 5 from hanging drop arrays, and placed on the top chamber of a transwell insert. The bottom chamber was filled with 400 μl of fresh medium, so only the bottom of the transwell insert was immersed in medium. Following 3 days, the transwell insert was removed, and several images of the bottom of the 24 well were obtained using phase contrast microscopy. Image J was used to quantify the number of cells in a field of view. At least four random non-overlapping fields of view were counted from each experiment, to find the number of cells that migrated through the transwell insert to the bottom of the well.

Hetero-spheroids were harvested and lysed in 200 μl of Radio-immunoprecipitation assay (RIPA) Buffer, sonicated for 30s on ice with a probe sonicator. Extracted concentration was measured using the BCA Assay Reagent (Pierce) following manufacturer’s protocol for a 96-well format. Subsequently, 50 μg of protein from each sample was loaded onto 4–20% gradient polyacrylamide gels (Biorad), and separated electrophoretically, transferred to a PVDF membrane. Transferred membranes were blocked with 5% non-fat milk, and probed with β-catenin (R&D Biosystems) overnight at 4 °C, washed with TBST buffer, and probed with an appropriate HRP-conjugated secondary antibody. β-Actin was used as a loading control to determine changes in β-catenin expression among samples. ECL reagent (Pierce Protein Biology) was used to visualize bands in a Biorad ChemiDoc Touch instrument. Digital images acquired were processed through NIH Image J, and band analysis tools were used for densitometry. Band densities were normalized against the loading control β-Actin, to determine changes.

Mission shRNA plasmids were obtained transformed into E. coli from Sigma Aldrich, targeted to WNT5B (TRCN0000123194). Transformed E. coli were grown in LB medium. Plasmid DNA was isolated using the Promega DNA isolation kit following manufacturer’s protocols, and 2.5 μg of DNA was transfected along with packaging pro-viral plasmids into HEK293-T cells. Lentivirus particles were isolated at 1X concentration by the University of Michigan Viral Vector Core. 1 × 10⁵ cells were transduced with 3 μg/ml polybrene and 0.5X lentivirus for 30 min at 800 g, at 32 °C in a centrifuge. Resulting pellets were re-suspended in fully supplemented growth medium for 72 h. At the end of 72 h, cells were harvested for qPCR analysis, or for macrophage differentiation and further experimentation. Lentiviruses were packaged to express shRNA targeting WNT5B (sh-WNT5B), or scrambled shRNA (sh-scramble). Sh-WNT5B or sh-scramble treated U937 monocytes were differentiated and activated into macrophages following protocols described in section 2.2.

CSC mono-spheroids and CSC/M2 and CSC/sh-WNT5B M2 hetero-spheroids were generated following protocols outlined in Section 2.4. CSCs were GFP tagged in these spheroids, and after five days of hetero-spheroid culture, CSCs were isolated using the GFP label prior to sub-cutaneous injection into NSG mice. Each tumor received CSCs from 10 spheroids. Tumor initiation and monitoring protocols were performed as described previously [13]. When palpable tumors were observed, tocilizumab (10 mg/kg, intra peritoneally) treatment began 3 times/week. Tumors were allowed to grow till the end-point was reached for maximum tumor burden, and mice were euthanized. Tumors were dissected, and routine paraffin histology and H&E staining was performed, to understand any changes in histology. RNA was isolated from tumors following protocols outlined in Section 2.6, and subject to qPCR for ALDH1A1, and several other Wnt ligands.

Experiments were carried out using 3–5 biological replicates for U937-derived macrophages, and OVCAR3-derived CSCs. GraphPad Prism 5.0 (www.graphpad.com) was utilized to perform all statistical analysis. When appropriate, one-way ANOVAs were used to test significant differences, and if differences were observed, indicated with symbols and a significance level.

The monocytic cell line, U937, or healthy-donor derived PBMCs were placed in hanging drop cultures. In the presence of PMA, U937 and PBMC monocytes differentiated into macrophages, and over the course of 72 h, they were organized as a compacted mass of cells within hanging drop cultures (Fig. 1a). Monocytes with no PMA stimulation, in contrast, were extremely loosely aggregated and did not form compact spheroids. Differentiated macrophages were indicated as M0 macrophages, implying differentiation with no cytokine stimulation. 74.6 ± 8.2% of U937 monocytes differentiated into M0 macrophages expressed the pan macrophage marker CD68. Similarly, PBMCs also differentiated into macrophages upon PMA stimulation, with 84.3 ± 8.5% CD68 expression (Fig. 1b). Monocytes were also differentiated and polarized into an alternatively activated phenotype (M2; Fig. 1a). These macrophages were derived with PMA stimulation in the presence of IL-4 and M-CSF. Alternately activated M2-like macrophages, either from U937 or PBMCs had an associated increase in gene expression of CD163 and CD206 (Fig. 1c). Immuno-suppressive cytokine IL-10 and tumor-promoting cytokine IL-6 were elevated in M2-polarized macrophages from both U937 and PBMC compared to M0 macrophages, or undifferentiated monocytes, indicating a shift in phenotype between M0 and M2-like macrophages derived using this culture system (Fig. 1d). In summary, macrophages could be derived from U937 or PBMC monocytes using the 3D hanging drop culture system, and could be further activated into a M2-phenotype. 3D differentiation and activation was similar in terms of gene expression to conventionally activated macrophages in 2D culture systems, including elevated arginase enzyme activity (Additional file 1: Figure S1).Fig1Fig. 1

Hetero-spheroids derived from monocyte-derived macrophages and ovarian cancer stem cells. a Monocytes from the U937 cell line or peripheral blood monocytes (PBMC) were plated into hanging drop arrays, and differentiated to M0 macrophages with phorbol ester treatment, or activated with IL-4 and MCSF treatment. Differentiated and activated macrophages formed compact spheroid-like aggregates. b Differentiated M0 and M2 macrophages all expressed pan macrophage marker, CD68 indicating 75–80% differentiation efficiency from monocytes to macrophages. CD68 expression was evaluated using flow cytometry analysis, with representative plots. c Polarization was assessed using qPCR analysis for two M2 genes, CD163 and CD206. Both U937 and PBMC macrophages expressed significantly higher levels of CD163 and CD206 genes, compared to untreated undifferentiated monocytes. d M2 differentiated macrophages secreted higher amounts of the immuno-suppressive cytokine IL-10, and the pro-tumoral cytokine IL-6. e CSCs were derived from the OVCAR3 cell line based on ALDH⁺ CD133⁺ expression. Hetero-spheroids were generated using differentiated U937 or PBMC M0 macrophages and CSCs or activated U937 or PBMC M2 macrophages and CSCs. Representative phase contrast images of hetero-spheroids seen at Day 5 following formation indicate compact spheroids, similar in size to CSC mono-spheroids generated from the same number of CSCs/spheroid. f Hetero-spheroids retain ~ 20% CD68 expression, indicating that at day 5, CD68⁺ macrophages constitute 20% of the population of cells. Scale bar = 200 μm

Hetero-spheroids combining ovarian CSCs derived from the OVCAR3 cell line and monocyte-derived macrophages (U937 or PBMC) in a starting ratio of 1:1 were generated using hanging drop arrays. Phase contrast images of hetero-spheroids at Day 5 indicate compact spheroid formation with tight defined boundaries (Fig. 1e). No significant differences in size or proliferative index were observed between CSC mono-spheroids, and CSC/M0 or CSC/M2 hetero-spheroids Additional file 1: Figure S2). Hetero-spheroids retained between 19.2 ± 1.5%-26.3 ± 1.8% expression of CD68⁺ macrophages at Day 5 (Fig. 1f). Gating strategy for flow analysis is presented in Additional file 1: Figure S3. CSC/M0 or CSC/M2 hetero-spheroids thereby maintained robust macrophage populations within the spheroids, while maintaining compact spheroid architecture for 5 days.

In order to understand if there are differences in bulk ovarian cancer cells (OVCAR3), and ovarian cancer stem cells (ALDH⁺ CD133⁺ CSC) in their ability to drive an immuno-suppressive macrophage phenotype, we generated hetero-spheroids from OVCAR3/M0 and CSC/M0. OVCAR3 or CSC mono-spheroids demonstrate minimal expression of CD206 (0.9–1%, Fig. 2a, Additional file 1: Figure S4). When comparing differences in CD206 expression between OVCAR3/M0 or CSC/M0 hetero-spheroids, a significant (**p < 0.001, one-way ANOVA) 20% increase in CD206 was observed with CSC co-culture (Fig. 2a), indicating that ovarian CSCs drive an immuno-suppressive phenotype in macrophages compared to bulk ovarian cancer cells. CSC/M2 hetero-spheroids also expressed another alternately activated M2 macrophage marker, CD163 (Additional file 1: Figure S5). We sought to explore whether there are differences in the immuno-suppressive cytokine IL-10 between OVCAR3 and CSC spheroids. We found that IL-10 gene expression was significantly elevated (2-fold) in CSC mono-spheroids compared to OVCAR3, which was even more pronounced in CSC/M0 spheroids compared to OVCAR3/M0 (Fig. 2b). In hetero-spheroids derived from M2 macrophages and CSCs, CD206 expression was maintained through co-culture (Fig. 2c). Higher secreted IL-10 levels were observed in CSC/M2 hetero-spheroids (Fig. 2d).Fig2Fig. 2

Hetero-spheroids drive CD206 polarization in monocyte-derived macrophages. a Hetero-spheroids were generated from bulk unsorted OVCAR3 cells and M0 macrophages (OVCAR3/U937 M0), and CSC/U937 M0. Flow analysis for the macrophage polarization marker CD206 indicated that while OVCAR3 and CSCs by themselves do not express CD206, CSCs drive CD206 expression in previously CD206⁻ M0 macrophages within hetero-spheroids at significantly (**p < 0.001, one-way ANOVA) higher levels than OVCAR3. Representative flow analysis plots are shown. b IL10 gene expression was evaluated using qPCR in mono- and hetero-spheroids, indicating that CSCs had a 2-fold increased gene expression of IL10, likely driving CD206 expression in M0 macrophages to higher extents than OVCAR3. c CD206 expression was maintained at elevated levels (18.5–29.24%) in all CSC hetero-spheroid conditions, indicating that the activated M2 phenotype was maintained in macrophages within hetero-spheroids. d Levels of secreted IL-10 were mildly elevated in CSC/M2 hetero-spheroids, but were also similar across all CSC/macrophage hetero-spheroids, indicating the presence of the immuno-suppressive cytokine. e When CSCs were pre-treated with the Wnt secretion inhibitor, IWP-2, significantly lower (*p < 0.05,one-way ANOVA) CD206 expression was observed in CSC/M2 hetero-spheroids, implying the importance of CSC-derived Wnt ligands in the maintenance of M2 activation in macrophages. f qPCR analysis revealed that gene expression of several Wnt ligands were elevated in CSC compared to bulk OVCAR3 spheroids (dotted line = no change); (g) Densitometric measurements of immunoblots for β-catenin compared to the loading control β-actin indicated that CSC/M2 spheroids had a 26% increase in β-catenin expression, compared to CSC mono-spheroids, indicating higher Wnt/β-catenin activity in CSC/M2 hetero-spheroids

We then investigated the maintenance of stemness within hetero-spheroids, since we hypothesized that alternative macrophage activation may result in a pro-tumoral reciprocity within hetero-spheroids. Flow analysis for elevated ALDH indicated that M2 hetero-spheroids significantly and robustly (**p < 0.001, one-way ANOVA) increased the maintenance of ALDH+ populations within hetero-spheroids, compared to CSC mono-spheroids (Fig. 3a). Macrophages themselves do not significantly express elevated ALDH in this assay (Additional file 1: Figure S6). A ~ 2 fold increase in ALDH⁺ populations was observed with co-culture with either U937 M2 or PBMC M2 hetero-spheroids, indicating that M2 activation resulted in improved ovarian CSC maintenance. Concomitant with this increase in ALDH, we also observed increased secretion of the IL-6 pro-tumoral cytokine (Fig. 3b). Consequently, inhibition of IL-6 signaling with two small molecule inhibitors, Ruxolitinib or SC144 during the formation of CSC/M2 hetero-spheroids significantly (**p < 0.001, one-way ANOVA) reduced the enrichment of ALDH⁺ cells (Fig. 3c). Our results indicate that the IL-6 signaling axis initiated by M2 macrophage co-culture significantly increases maintenance of ALDH⁺ CSCs within hetero-spheroids.Fig3Fig. 3

M2-polarized macrophages increase ALDH+ in CSC/M2 hetero-spheroids. a Elevated ALDH activity was assessed using the ALDEFLUOR flow cytometric analysis assay, with representative flow plots demonstrated. CSC/M2 hetero-spheroids, whether U937 or PBMC, have significantly (**p < 0.001, one-way ANOVA) elevated ALDH⁺ expression (1.8–2.4 fold). b Secreted pro-tumoral cytokine IL6 is elevated in hetero-spheroids compared to CSC mono-spheroids. c We suppressed signaling through the IL6/STAT3 axis using two inhibitors Ruxolitinib and sc144 in hetero-spheroids, and observed that this suppression also significantly (**p < 0.001, one-way ANOVA) decreased the ALDH⁺ enrichment within CSC/M2 hetero-spheroids. d Concomitant with the ALDH expression within hetero-spheroids, sensitivity to carboplatin was evaluated using the MTS assay. Normalized absorbance values indicated that CSC/M2 hetero-spheroids were significantly (*p < 0.05, one-way ANOVA) more resistant to carboplatin treatment compared to CSC mono-spheroids made with the same number of CSCs. e Representative phase contrast images indicate the loss of the tight spheroid boundaries in the CSC mono-spheroids in response to carboplatin treatment, indicative of cell death. The extent of the loss of boundary integrity is much lower in the CSC/M2 hetero-spheroids. Scale bar = 100 μm. f Quantification of the number of migrated cells in the lower chamber of a transwell system indicated that CSC/M2 hetero-spheroids were significantly (*p < 0.05, one-way ANOVA) more migratory compared to CSC mono-spheroids

We hypothesized that similar to CSCs, M2-like macrophage activation may also result in increased WNT signaling. We blocked pan-WNT ligand secretion in M2 macrophages by treatment with IWP-2 following polarization. CSC/IWP2 M2 hetero-spheroids assumed different aggregation morphology and were less compact (Fig. 4a). Furthermore, ALDH expression was significantly lower (**p < 0.001, one-way ANOVA) in CSC/IWP2 M2 hetero-spheroids compared to CSC/M2, with minimal reduction in CD206 expression (Fig. 4b), indicating that macrophage-derived WNT signaling may at least be partially responsible for the phenotypes observed in ovarian CSCs. We investigated the expression of WNT ligands in macrophages, and found WNT5B over-expressed ~ 32 fold in M2 macrophages compared to monocytes using qPCR. We used an shRNA plasmid targeting the WNT5B ligand (sh-WNT5B) and a non-targeted plasmid (sh-Scramble), and used lentiviral methods to silence WNT5B expression in monocytes, with a knockdown efficiency of 76% (Fig. 4c). Macrophages derived from sh-WNT5B were termed sh-WNT5B M2. Despite > 75% knock-down efficiency of WNT5B in monocytes, Alternate M2-like activation still resulted in an increase in gene expression of WNT5B. However, we found that monocytes treated with sh-WNT5B showed a 52% reduction in WNT5B expression compared to control or scramble M2 macrophages (Fig. 4d). WNT5B knockdown did not alter the CD68⁺ marker expression (Fig. 4e). Treatment with IL-4/M-CSF to induce macrophage polarization resulted in increased gene expression for CD163 and CD206, indicating the development of an alternate M2-like phenotype (Fig. 4f). Therefore, knockdown of WNT5B using sh-WNT5B did not significantly alter the differentiation of monocytes to macrophages, or the development of an M2 activated phenotype. However, a significant loss in WNT5B gene expression was observed.Fig4Fig. 4

Inhibition of macrophage Wnt-secretion reduces ALDH enrichment in hetero-spheroids. a Representative phase contrast image of a hetero-spheroid generated from CSCs and IWP-2 treated U937 M2 macrophages, indicates the formation of aggregated spheroids. Scale bar = 200 μm. b Flow analysis revealed that hetero-spheroids with IWP-2 treated M2 macrophages had a significantly diminished ALDH+ enrichment (**p < 0.001, one-way ANOVA) compared to control untreated CSC/M2 hetero-spheroids. However, no change in CD206 expression was observed in hetero-spheroids. c Transduction efficiency of monocytes with shRNA directed against WNT5B indicated a > 75% efficiency in knockdown of WNT5B gene expression in the shWnt5b targeted construct, and minimal changes in WNT5B gene expression in the scramble non-targeted lentiviral construct. d We utilized shWnt5b monocytes to differentiate and polarize M2 macrophages, and found that there was a 52% reduction in WNT5B gene expression in shWnt5b M2 macrophages, compared to scramble or control untreated M2 macrophages, indicating the knockdown of the Wnt5b gene. e No changes were observed in CD68 expression in monocytes generated from scramble or shWNT5B treated monocytes, demonstrated by flow analysis and representative plots. f Similarly, qPCR analysis of M2 gene expression markers CD163 and CD206 indicated increases in both genes, indicative of activation into the M2 program even in shWnt5b macrophages

Hetero-spheroids were generated with CSCs and Scramble M2 or sh-WNT5B M2. No significant differences in CD206 expression was observed in these hetero-spheroids, indicating that in co-culture, macrophage knockdown of WNT5B was dispensable for the expression of CD206 (Fig. 5a). However, macrophage knockdown of WNT5B resulted in a significant (*p < 0.05, one-way ANOVA) decrease in ALDH⁺ compartment by over 5-fold, indicating that macrophage-derived WNT5B was crucial for the enrichment of CSC characteristics within hetero-spheroids (Fig. 5b). This reduction in the ALDH⁺ enrichment was also associated with an increased sensitivity to carboplatin (Fig. 5c), and a decreased invasive potential (Fig. 5d). Upon examination of cytokine profiles, we observed no significant differences in IL-10 (Fig. 5e). However, the pro-tumoral CSC-promoting cytokine IL-6 was completely abrogated in the CSC/sh-WNT5B M2 hetero-spheroids, indicating that WNT5B was likely also driving CSC phenotypes through IL-6 secretion. We explored the possibility of restoring the CSC enrichment phenotype by the addition of exogenous IL-6. Upon exogenous addition of IL-6, levels of ALDH⁺ cells increased within CSC/sh-WNT5B M2 hetero-spheroids, indicating that IL-6 is a key effector downregulated upon WNT5B knockdown, but may likely not be the only mediating factor in promoting CSC maintenance via WNT5B (Fig. 5f). Concomitant with the loss of IL-6 in CSC/sh-WNT5B M2 hetero-spheroids, we also observed reduced phosphorylated STAT3 (42.4 ± 5.5%) in immunoblots compared to CSC/M2 hetero-Additional file 1: Figure S7). Lastly, we explored the possibility of WNT-driven WNT signaling in CSCs, in response to macrophage WNT5B. We observed through gene expression analysis that several WNT ligands were significantly over-expressed in CSCs co-cultured with M2 macrophages, and there was a loss in WNT ligand expression upon co-culture with sh-WNT5B M2 macrophages (Fig. 5g). Consequently, there was also an associated ~ 50% loss of β-catenin protein expression in CSC/sh-WNT5B M2 hetero-spheroids, indicating a lower paracrine WNT activation in CSCs in co-culture with macrophages where WNT5B was knocked down (Fig. 5h).Fig5Fig. 5

Macrophage knockdown of Wnt5b suppresses ALDH enrichment, with no change in CD206, and increases chemosensitivity of hetero-spheroids. a Flow analysis of CD206 indicated no significant differences in CSC/M2 and CSC/Scramble M2 and CSC/shWnt5b M2 hetero-spheroids, indicating that CSCs were still capable of maintaining a robust immunosuppressive program in hetero-spheroids, regardless of WNT5B. b ALDH elevation however was significantly (*p < 0.05, one-way ANOVA) lowered compared to CSC/U937 M2 hetero-spheroids, and not significantly different (ns) compared to CSC mono-spheroids, indicating that knocking down Wnt5b expression in macrophages reduced ALDH enrichment in hetero-spheroids. c Concomitant with the decrease in ALDH, sensitivity to carboplatin also significantly (**p < 0.001, one-way ANOVA) improved, responding to treatment similar to CSC mono-spheroids. The red dashed line indicates the sensitivity levels of CSC mono-spheroids. d CSC/shWnt5b M2 hetero-spheroids were also significantly (***p < 0.0001, t-test) less invasive than CSC/Scramble M2 hetero-spheroids. e Upon ELISA analysis of secreted IL10 and IL6, we found no changes in IL-10 levels, while we found a stark decrease in secreted pro-tumoral IL-6 cytokine in CSC/shWnt5b M2 hetero-spheroids. f Exogenous addition of IL-6 (25 ng/ml) to CSC/shWnt5b M2 hetero-spheroids partially significantly (*p < 0.05, one-way ANOVA) increases ALDH+ enrichment, but does not restore it to levels of original enrichment with CSC/Scramble M2 or CSC/Ctrl M2 hetero-spheroids. g In order to identify if there was paracrine macrophage Wnt-driven Wnt signaling in CSCs, we separated the CSCs (using a GFP label) from hetero-spheroids, and probed for gene expression of several Wnt ligands we saw elevated in CSCs compared to bulk OVCAR3. We observed that with M2 macrophage co-culture, several Wnt ligands were upregulated (Wnt2 significantly elevated, ***p < 0.001, two-way ANOVA). However, this elevated gene expression of Wnt ligands was lost in CSCs in hetero-spheroid culture with shWnt5b M2, indicating that Wnt5b was partly responsible for potentiating Wnt signaling within the CSC compartment. h This loss in Wnt ligand expression also translated to decreased β-catenin expression in CSC/shWnt5b M2 hetero-spheroids compared to CSC/U937 M2 hetero-spheroids, quantified by densitometry of immunoblots (representative blot shown)

Three tested conditions: CSC (mono-spheroids), CSC (M2; conditioned in hetero-spheroids) and CSC(sh-WNT5B M2; conditioned in hetero-spheroids) generated tumors in NSG mice (Fig. 6a). The rate of tumor initiation and kinetics of growth was significantly different between all three conditions, with CSC(M2) tumors initiating at the fastest rate of 11.97mm³/day. CSC(shWNT5b M2) tumors were significantly slower to initiate at 4.74mm³/day, compared to CSC tumors at 5.89mm³/day. CSC(M2) tumors reached the maximum tumor burden window earlier, compared to CSC and CSC(shWNT5B M2) tumors (Fig. 6a). Histologic examination of the xenografts reveals that the lesions are composed of solid sheets of tumor cells with epithelioid morphology. In the CSC group, dense sheets of polygonal tumor cells with abundant cytoplasm and conspicuous nuclei are noted, with little intercellular space (Fig. 6b). In the CSC(M2) group, sheets of large epithelioid tumor cells with abundant eosinophilic cytoplasm constitute the entire tumor with little intercellular space and stroma. In the CSC(shWNT5B M2) group, the tumor cells are much smaller and loosely arranged in cords and strands. Occasional apoptotic cells are seen with a low mitotic count. Gene expression levels were assessed in xenografted tumors from the three groups, to understand if macrophage conditioning was maintained in vivo (Fig. 6c). Our data indicated that an elevated ALDH1A1 expression was retained significantly in CSC(M2) tumors (**, p < 0.001, two-way ANOVA), compared to CSC(shWNT5B M2) tumors (ns; not significant). Similarly, elevated Wnt ligands observed in vitro upon CSC/macrophage co-culture in hetero-spheroids (Fig. 5g) was re-evaluated in xenografts. We observed the maintenance of a significant elevation in Wnt2, Wnt3A, and Wnt6 (*p < 0.05, **p < 0.001, two-way ANOVA) in CSC(M2) tumors compared to CSC tumors. CSC(shWNT5B M2) tumors were not significantly different in Wnt ligand elevation compared to CSC tumors. Furthermore, we tested the effect of the human IL-6 inhibitor Tocilizumab on CSC and CSC(M2) spheroids (Fig. 6d). CSC spheroids were significantly responsive to Tocilizumab treatment, evident from the divergent treatment curve from the control untreated group (purple curves, Fig. 6d). CSC(M2) tumors were however significantly resistant to Tocilizumab treatment, indicating the elevated IL6 conditioning activity, in line with elevated ALDH1A1 gene expression and elevated Wnt ligand expression. Change in tumor burden with Tocilizumab treatment in CSC spheroids ranged between 67.7–70.7%, while in CSC(M2) tumors, the reduction was merely 23.8–24% of control tumor volumes.Fig6Fig. 6

Changes in tumorigenicity of hetero-spheroids in vivo in NSG mice. Tumors were generated in NSG mice from CSC mono-spheroids, or CSCs isolated from hetero-spheroids in CSC/M2 or CSC/shWNT5b M2. a Tumor initiation and growth kinetics are shown for all three groups, where CSC(M2) tumors (red curve) demonstrates elevated tumorigenicity, with faster tumor initiation and tumor burden development (gray shaded area indicates the tumor burden window). Similarly, the blue trace indicates the tumor growth in CSC/shWNT5b M2 tumors, which is significantly (*p < 0.05, two-way ANOVA) lower and slower compared to CSC and CSC(M2) groups at the time points indicated. b Histological examination demonstrates that all groups establish similar structures of tumors sub-cutaneously, with CSC tumors and CSC (M2) tumors establishing dense epitheliod cells, with CSC (shWNT5b M2) tumors more loosely arranged. Scale bar = 100 μm. c Gene expression analysis of xenografted tumors indicated that an elevated ALDH1A1 expression was maintained in CSC(M2) tumors (**p < 0.001, two-way ANOVA). M2-like macrophage conditioning also helped CSCs retain elevated expression of WNT2, WNT3A and WNT6 (*p < 0.05, **p < 0.001, two-way ANOVA) compared to CSCs in mono-spheroids. All other genes and conditions were not significantly different (ns) compared to CSCs, specifically CSC/shWnt5B M2 tumors. d The human IL-6 inhibitor Tocilizumab was used intra-peritoneally on CSC and CSC(M2) tumors, once palpable tumors were observed (Day 25), indicated by the arrow. Tocilizumab treatment resulted in a significant decrease in tumor volume (**p < 0.001, two-way ANOVA) compared to control untreated tumors in CSC mono-spheroid tumors (compare purple trace to black trace; ~ 70% reduction in tumor burden). However, in CSC/M2 tumors, tocilizumab treatment did not significantly alter the growth kinetics of treated vs. untreated tumors (~ 24% reduction in tumor burden), indicating the detainment of a chemoresistant phenotype upon M2-like macrophage co-culture and conditioning. Furthermore, the treated CSC Tocilizumab condition also resembles the control CSC (shWNT5B M2) tumors (blue trace in A), indicating that partial loss or inhibition of IL-6 based CSC enrichment may be at play