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. 2023 Jun 19:13:1192472.
doi: 10.3389/fonc.2023.1192472. eCollection 2023.

Tocilizumab (monoclonal anti-IL-6R antibody) reverses anlotinib resistance in osteosarcoma

Jiuhui Xu  1   2 , Chenglong Chen  2   3 , Kunkun Sun  4 , Qianyu Shi  1   2 , Boyang Wang  1   2 , Yi Huang  1   2 , Tingting Ren  1   2 , Xiaodong Tang  1   2
Affiliations

Tocilizumab (monoclonal anti-IL-6R antibody) reverses anlotinib resistance in osteosarcoma

Jiuhui Xu et al. Front Oncol. .

Abstract

Purpose: Anlotinib, a tyrosine kinase inhibitor (TKI) has been in clinical application to inhibit malignant cell growth and lung metastasis in osteosarcoma (OS). However, a variety of drug resistance phenomena have been observed in the treatment. We aim to explore the new target to reverse anlotinib resistance in OS.

Materials and methods: In this study, we established four OS anlotinib-resistant cell lines, and RNA-sequence was performed to evaluate differentially expressed genes. We verified the results of RNA-sequence by PCR, western blot and ELISA assay. We further explored the effects of tocilizumab (anti- IL-6 receptor), either alone or in combined with anlotinib, on the inhibition of anlotinib-resistant OS cells malignant viability by CCK8, EDU, colony formation, apoptosis, transwell, wound healing, Cytoskeletal stain assays, and xenograft nude mouse model. The expression of IL-6 in 104 osteosarcoma samples was tested by IHC.

Results: We found IL-6 and its downstream pathway STAT3 were activated in anlotinib-resistant osteosarcoma. Tocilizumab impaired the tumor progression of anlotinib-resistant OS cells, and combined treatment with anlotinib augmented these effects by inhibiting STAT3 expressions. IL-6 was highly expressed in patients with OS and correlated with poor prognosis.

Conclusion: Tocilizumab could reverse anlotinib resistance in OS by IL-6/STAT3 pathway and the combination treatment with anlotinib rationalized further studies and clinical treatment of OS.

Keywords: anlotinib; drug resistance; interleukin-6; osteosarcoma; tyrosine kinase inhibitor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The validation of established anlotinib-resistant OS cells. (A) The results of CCK8 assay showed IC50 values of KHOS cells and KHOS-R were13.91μM and 26.38μM respectively. (B) The IC50 value of 143B cells to anlotinib is 4.758μM, and 143B-R cells is 15.25 μM. (C) The colony formation results indicated KHOS-R and 143B-R were more insensitive to anlotinib. (D) The quantified data of colony formation assay. (E) The results of apoptosis showed both KHOS-R and 143B-R cells were more insensitive to anlotinib for 24h treatment. (***P < 0.001, ****P < 0.0001).
Figure 2
Figure 2
IL-6/STAT3 axis played a critical role in OS anlotinib resistance. (A) The heatmap showed the differentially-expressed genes between NC (OS cell lines) and RE (anlotinib-resistant cell lines). IL-6 was up-regulated in anlotinib-resistant cells (143B-R, KHOS-R, MG63-R, U2OS-R). (B) KEGG enrichment analysis showed the differentially-expressed enrichment signaling pathways between OS cell lines and anlotinib-resistant OS cells. Cytokine-cytokine receptor interaction was highly active in anlotinib-resistant OS cells (143B-R, KHOS-R, MG63-R, U2OS-R). (C) Gene set enrichment analysis (GSEA) validated enrichment of cytokine activity in anlotinib-resistant OS cell lines. (D) The volcano plot showed IL-6 was up-regulated in anlotinib-resistant OS cell lines. (E) The qPCR results showed IL-6 mRNA expression was both increased in KHOS-R and 143B-R cells when compared with OS cell lines. (F) The ELISA assay results showed IL-6 secretion was both increased in KHOS-R and 143B-R cells when compared with OS cell lines. (G) Western blot results indicated IL-6 protein expression was up-regulated in 143B-R and KHOS-R cells. (H) The results of Western blot showed STAT3 protein was increased in anlotinib resistant cells which revealed STAT3 pathway was activated. (**P <0.01, ****P < 0.0001).
Figure 3
Figure 3
Combination with tocilizumab inhibited the proliferation of anlotinib-resistant cells. (A) The IC50 values of combination treatment were decreased to 12.70μM and 9.388μM from 23.76μM and 16.91μM for KHOS-R and 143B-R cells respectively. (B) The number of colony formation was significantly inhibited in combination treatment group of anlotinib resistant cells, and tocilizumab monotherapy showed anti-tumor function on 143B-R cells. (C, D) The statistical analysis of colony formation results of KHOS-R and 143B-R cells. (E) Representative pictures from Edu assay. The results showed combination treatment obviously inhibited the proliferation viability of KHOS-R cells. (F, G) The results of apoptosis assay showed combination treatment obviously promoted anlotinib-resistant OS cells apoptosis. (ns, no significance; *P<0.05, **P<0.01, ****P<0.0001).
Figure 4
Figure 4
Combination therapy was more effective to suppress OS malignant progression by blocking IL-6/STAT3 axis. (A) The migration ability of anlotinib-resistant OS cells was significantly inhibited by the combination treatment with tocilizumab. (B, C) The statistical plots showed the combination treatment with tocilizumab decreased the healing rate of anlotinib-resistant cells, and migration ability of anlotinib-resistant KHOS cells could also be inhibited by tocilizumab monotherapy. (D) The results of transwell assay showed the migration ability of anlotinib-resistant OS cells was significantly inhibited by the combination treatment. (E) The statistical data of transwell assay for 143B-R and KHOS-R cells. (F) Western blot results showed the EMT-related protein N-cad expression and vimentin expression were decreased which was caused by combination treatment. (G) The combination treatment with tocilizumab inhibited cytoskeleton formation of anlotinib-resistant OS cells. (H) Western blot results showed the STAT3 expression and phosphorylation were inhibited because of the combination treatment. (ns, no significance; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 5
Figure 5
Tocilizumab enhanced the antitumor effect of anlotinib in vivo. (A) Xenograft photographs were taken after 14-day administration with anlotinib monotherapy, tocilizumab monotherapy and combination therapy. (B) Growth curves of tumors presented by volume evaluated once 4 days. (C) The results of tumor weights at the day of mice were euthanized. (D) Representative images of lungs harvested from mice after 14 days of treatment. (E) The statistical analysis results of pulmonary metastasis. (F) Representative STAT3 staining of tumor sections from mouse models were shown. (G) Quantification of STAT3 expression was performed in different treatment groups. (ns, no significance; *P < 0.05).
Figure 6
Figure 6
IL-6 was highly expressed in patients with osteosarcoma and correlated with poor prognosis. (A) Total 104 tissues from OS patients were collected and IL-6 expression was shown by IHC. (B) The IRS score of IL-6 expression was determined and translated into three levels: high (IRS 9–12), medium (IRS 5–8), and low (0–4). (C) The survival analysis indicated the patients with high level IL-6 expression were correlated with poor prognosis (n=104). (D) Schematic diagram illustrating the hypothetic mechanisms of combination therapy with tocilizumab enhances sensitivity to anlotinib for anlotinib resistant OS cells.
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