Cancer-associated mesenchymal stroma fosters the stemness of osteosarcoma cells in response to intratumoral acidosis via NF-κB activation

Abstract

The role of mesenchymal stem cells (MSC) in osteosarcoma (OS), the most common primary tumor of bone, has not been extensively elucidated. We have recently shown that OS is characterized by interstitial acidosis, a microenvironmental condition that is similar to a wound setting, in which mesenchymal reactive cells are activated to release mitogenic and chemotactic factors. We therefore intended to test the hypothesis that, in OS, acid-activated MSC influence tumor cell behavior. Conditioned media or co-culture with normal MSC previously incubated with short-term acidosis (pH 6.8 for 10 hr, H⁺ -MSC) enhanced OS clonogenicity and invasion. This effect was mediated by NF-κB pathway activation. In fact, deep-sequencing analysis, confirmed by Real-Time PCR and ELISA, demonstrated that H⁺ -MSC differentially induced a tissue remodeling phenotype with increased expression of RelA, RelB and NF-κB1, and downstream, of CSF2/GM-CSF, CSF3/G-CSF and BMP2 colony-promoting factors, and of chemokines (CCL5, CXCL5 and CXCL1), and cytokines (IL6 and IL8), with an increased expression of CXCR4. An increased expression of IL6 and IL8 were found only in normal stromal cells, but not in OS cells, and this was confirmed in tumor-associated stromal cells isolated from OS tissue. Finally, H⁺ -MSC conditioned medium differentially promoted OS stemness (sarcosphere number, stem-associated gene expression), and chemoresistance also via IL6 secretion. Our data support the hypothesis that the acidic OS microenvironment is a key factor for MSC activation, in turn promoting the secretion of paracrine factors that influence tumor behavior, a mechanism that holds the potential for future therapeutic interventions aimed to target OS.

Keywords: cancer stemness; inflammation; mesenchymal stroma; tumor microenvironment.

Figure 1. Morphology and immunostaining of canine OS tissue sample

A red asterisk (*) indicates the same area in all the histological sections and includes a borderline zone between the tumor area and the periosteal stromal reaction. (A) Histological sample of canine OS stained by standard hematoxylin and eosin (H & E). Scale bar 500 µm; (B) Immunohistochemistry for vimentine, α-SMA, and Ki67 of the same sections shown in panel A. For vimentin, both high and low magnifications are shown. Arrows indicate positive staining. Scale bar 200 µm; (C) Cell cultures of normal human MSC, of T-MSC derived from a human OS tissue samples, and OS cells (HOS) stained for the same markers shown in panel B. Scale bar 200 µm.

Figure 2. Short-term acidosis induces NF-κB activation in MSC

(A) mRNA expression of NF-κB transcription factors in MSC (n=3, 2 from bone marrow and 1 from adipose tissue) exposed to acidic medium at different times. Mean ± SE (n=12), ***P<0.001, and *P<0.05; (B) Quantification of the signal for NF-κB transcription factors translocated into the nucleus of MSC (n=3) after 24 hrs of culture with medium at different pH. Mean ± SE (n=12); ***P<0.001, **P<0.01, and *P<0.05; (C) Heat map representation of the fold increase, by deep-sequencing analysis, of the mRNA levels of NF-κB-related genes of OS cells (MG63, HOS, Saos-2) and normal stromal cells (2 lots of skin fibroblasts, FB, and 4 lots of BM-MSC) after short-term acidosis (pH 6.5) compared to physiological medium (pH 7.4). Colors on the heat map indicate the log2 ratios of expression (representing normalized read counts). Red, upregulation; green, downregulation. (D) Clustering in gene categories of the results obtained by the deep-sequencing analysis of the transcriptional levels of NF-κB-related genes. Mean ± SE; *P<0.05. (E) LAMP2 staining of histological sample of canine OS and an indirect marker of acidic area in the tumor microenvironment; the same sections was also stained for (F) NF-κB p65 (RelA, B1 scale bar 100 µm) also shown at higher magnification (scale bar 50 µm) (G) and NF-κB (G). Arrows indicate positive staining.

Figure 3. Short-term acidosis promotes the release of the inflammatory cytokines IL6 and IL8 by the tumor-associated mesenchymal stroma

(A) Human Inflammatory Cytokines Multi-Analyte ELISArray™ performed on MSC cultured in acidic or physiological medium for 24 hours (n=3); (B) IL6 and IL8 protein expression in the same samples after 24 hrs from the start of the incubation with acidic medium by using specific ELISA. Mean ± SE (n=12), *P<0.05; (C) IL6 and IL8 mRNA analysis of MSC at different time points after the incubation with acidic medium. Mean ± SE (n=12), ***P<0.001, **P<0.01, and *P<0.05 vs T0; (D) The induction of IL6 and IL8 mRNA expression by the acidic medium is reversible: mRNA levels of IL6 and IL8 were analyzed at T0, when the medium was change with acidic medium, at T1 at the end of the incubation period, and at T2, after additional 16 hours from the interruption of the acidic stimulus. Mean ± SE of each different lot of MSC (n=12), CSF2/GM-CSF, CSF3/G-CSF, BMP2 mRNA analysis of MSC after 6 hours of culture in acidic medium. Mean ± SE (n=12); (E) IL8 serum level trend in patients at diagnosis and at 12 months since diagnosis (after treatment).

Figure 4. Short-term acidosis induces a MSC-derived secretome that boosts the migration and invasive potential and the colony formation of OS cells

(A) CSF2/GM-CSF, CSF3/G-CSF, BMP2 mRNA analysis of MSC after 6 hours of culture in acidic medium. Mean ± SE (n=12); ***P<0.001, **P<0.01, and * P<0.05 vs pH 7.4; (B) Clonal efficiency of MSC in acidic medium. Mean ± SE (n=18); *P<0.05 vs pH 7.4; (C) Clonal efficiency of OS cells when cultured in CM of MSC that were pre-treated (CM MSC^{pH 6.8}) or not (CM MSC^{pH 7.4}) with acidic medium. Mean ± SE (n=12), ***P<0.001, **P<0.01 vs CM MSC^{pH 7.4}. (D) CCL5, CXCL5, CXCL1, and CXCR4 mRNA analysis of MSC after 6 hours of culture in acidic medium. Mean ± SE (n=12), ***P<0.001, **P<0.01 vs pH 7.4; (E) Migration of MSC and HOS cells and (F) invasion ability of HOS cells exposed to CM of MSC pre-treated (CM MSC^{pH 6.8}) or not (CM MSC^{pH 7.4}) with acidic medium that here were used as chemotactic stimulus. Mean ± SE (n=3). In the migration assay the right panels are representative images of the porous membrane with migrated cells stained with crystal violet (for MSC 10× lens, for HOS 20× lens), whereas in the invasion assay the same type of images are on the left panel (20× lens). *P<0.05 vs CM MSC^{pH 7.4} (G) MMP11 mRNA analysis of MSC after 6 hrs of culture in acidic medium. Mean ± SE (n=12); ***P<0.001 vs pH 7.4. (H) MMP11 protein expression in supernatant of MSC cultured or not in acidic medium for 24 hrs. Mean ± SE (n=12).

Figure 5. The secretome of acid-stressed MSC promote a stem-like phenotype in HOS cells

(A). Sphere-forming efficiency assay in HOS cells co-cultured with MSC that were previously stressed or not with acidic medium. Left panel, representative images of the obtained spheres (4× lens). Mean ± SE (n=9), *P<0.05; (B) mRNA expression of the stem-related genes OCT4 and Nanog in HOS co-cultured for 6 days with MSC that were pretreated (CM MSC^{pH 6.8}) or not (CM MSC^{pH 7.4}) with acidic medium. Mean ± SE (n=4), *P<0.05; (C) Sphere-forming efficiency (left panel) and stem-related gene expression (right panel) of HOS cells treated with IL6 and IL8. Mean ± SE (n=3), *P<0.05; (D) Sphere-forming efficiency of HOS co-cultured for 6 days with MSC that were pretreated (CM MSC^{pH 6.8}) with acidic medium with or w/o Tocilizumab (anti-IL6 antibody). Mean ± SE (n=3), *P<0.05; (E) Indirect viability assay (left panel) and apoptosis assay (right panel) to measure the effect of CM obtained from MSC treated with acidic medium (MSC CM^{pH 6.8}) on HOS resistance to doxorubicin (DXR). Mean ± SE (n=8 for viability test and n=6 for apoptosis test), *P<0.05 and **P<0.01 vs DXR, ^§§P<0.01 vs CM MSC^{pH 7.4}; (F) Due to the high glycolytic metabolism, OS cells strongly acidify the TME. The local increased concentration of protons is sensed by MSC in the TME as a strong exogenous stress. The acid-stressed mesenchymal stroma might be therefore reprogrammed into T-MSC by the activation of the NF-κB inflammatory pathway that triggers the transcription and the secretion of several cytokines and chemomikes, among which IL6, IL8, CCL5, GM-CSF, CXCL1, and CXCL5. Through a paracrine circuit, the secretome derived from the reactive mesenchymal stroma, in turn, might promote the stemness and migration of OS cells.