Drug-induced senescence by aurora kinase inhibitors attenuates innate immune response of macrophages on gastric cancer organoids

Abstract

Diffuse-type gastric cancer (DGC) is a subtype of gastric cancer with aggressiveness and poor prognosis. It is of great significance to find sensitive drugs for DGC. In the current study, a total of 20 patient-derived organoids (PDOs) were analyzed for screening the therapeutic efficacy of small molecule kinases inhibitors on gastric cancers, especially the therapeutic difference between intestinal-type gastric cancer (IGCs) and DGCs. The IGCs are sensitive to multiple kinases inhibitors, while DGCs are resistant to most of these kinases inhibitors. It was found that DGCs showed drug-induced senescent phenotype after treatment by aurora kinases inhibitors (AURKi) Barasertib-HQPA and Danusertib. The cell diameter of cancer cells are increased with stronger staining of senescence-associated β-galactosidase (SA-β-GAL), and characteristic appearance of multinucleated giant cells. The senescent cancer cells secrete large amounts of chemokine MCP-1/CCL2, which recruit and induce macrophage to M2-type polarization in PDOs of DGC (DPDOs)-macrophage co-culture system. The up-regulation of local MCP-1/CCL2 can interact with MCP-1/CCL2 receptor (CCR2) expressed on macrophages and suppress their innate immunity to cancer cells. Overall, the special response of DGC to AURKi suggests that clinicians should select a sequential therapy with senescent cell clearance after AURKi treatment for DGC.

Keywords: AURK; Drug screening; Gastric cancer; Immunosuppressive; Patient-derived organoids.

Fig. 1

Drug sensitive screening using a panel of kinases inhibitors on IPDOs and DPDOs. (a) Flow chart of drug screening for kinases inhibitors in PDOs of GC. (b) A panel of kinases inhibitors, CAS number, and cell vitality of PDOs are presented. (c) The GSVA pathway analysis for IPDOs and DPDOs. (d) The heatmap shows sensitivity difference between IPDOs and DPDOs to kinases inhibitors. The color column on the right side represents drug sensitivity. The red indicates sensitivity and the blue indicates resistance. (e) The bar chart shows cell vitality difference to Barasertib-HQPA between IPDOs and DPDOs (**P = 0.0013). (f) The bar chart shows cell vitality difference to Danusertib between IPDOs and DPDOs (**P = 0.0018). (g) The transcriptomic levels of AURKB gene in IPDOs and DPDOs (**P = 0.002). (h) The correlation analysis of AURKB level and cell vitality upon Barasertib-HQPA treatment (r = −0.7658). (i) The correlation analysis of AURKB level and cell vitality upon Danusertib treatment (r = −0.6185).

Fig. 2

The specific response of DPDOs to AURKi Barasertib-HQPA and Danusertib. (a) In HE staining of PDOs slice, four DPDOs shows the morphology of DGC without obvious glandular structure and poorly adhesive arrangement. (b) Compared to NC group, the cell diameter of Danusertib group or Barasertib-HQPA group is significantly increased, with no cell lysis and necrosis. (c) By Danusertib and Barasertib-HQPA treatment (1 μΜ, 96 h), the cell diameter of four DPDOs is significantly increased compared to NC group, n = 10，scale bar = 30 μm; **P < 0.01; ***P < 0.001.

Fig. 3

Drug-induced cellular senescence in DPDOs with up-regulation of MCP-1/CCL2. (a) The positive rate of SA-β-GAL staining is increased in both DPDO1 and DPDO2 after Danusertib or Barasertib-HQPA treatment (1 μM，96 h). n = 10，scale bar = 30 μm. (b) Compared to NC group, enlarged cancer cells with multinucleated giant cell changes are observed in Danusertib or Barasertib-HQPA treated group by Giemsa staining. scale bar = 20 μm (c) The bar chart shows increased positive rate of SA-β-GAL in six aging PDOs relative to eight early passage PDOs. (d) The heatmap shows inflammatory factors concentration of culture supernatants from active PDOs and aging PDOs. The red box marks the up-regulation of MCP-1/CCL2. (e) The bar chart presents the concentration difference of MCP-1/CCL2 between aging PDOs and active PDOs. (f) The bar chart shows concentration difference of MCP-1/CCL2 in the culture supernatants of NC group and Danusertib or Barasertib-HQPA-treated groups (1 μM, 96 h) in DPDO1 and DPDO2 cases. n = 3; ^nsP> 0.05; **P < 0.01; ***P < 0.001.

Fig. 4

The relationship between cell types and the expression of MCP-1/CCL2 in scRNA-seq analysis. (a) In tSNE plot of scRNA-seq analysis, the cells were divided into eleven clusters, including epithelial cells (red line marked) and macrophage (blue line marked). (b) Using KRT18 as the epithelial marker gene, the epithelial cells are further divided into seven clusters (C0 to C6). (c) Senescence associated genes of CDKN2A and CDKN2B are mainly expressed in the C3 cluster. (d) Among C0 to C6 clusters, the proportion of MCP-1/CCL2 expressing cells is the highest (7.11 %) in the C3 cluster. (e) In GSEA analysis, up-regulated genes of C3 are enriched in pathways of reactive oxygen species，mTORC1 signaling，IL6-JAK-STAT3 signaling，and so on. (f) Using CSF1R as the marker gene, the macrophages are further divided into six clusters (C0 to C5). (g) In violin plot, the M1 polarization cells (C4) express high levels of CD80 and CD86, while the M2 polarization cells (C0) express high levels of CD206 and CD163. (h) The gene expression feature of MCP-1/CCL2 in macrophages. (i) Among six clusters of macrophages, the proportion of MCP-1/CCL2 expressing cell is the highest (17.64 %) in the C0 cluster. (j) By GSEA analysis, the up-regulated genes of C0/M2 cluster are enriched in pathways of TNFα signaling via NF-κB，inflammatory response，complement，and so on.***P < 0.001.

Fig. 5

Up-regulation of MCP-1/CCL2 mediates immunosuppressive effect on DPDOs-macrophage co-culture system. (a) The bar chart shows concentration of MCP-1/CCL2 in supernatants of M0, M1, and M2 macrophages. n = 3. (b) The migrated cells of macrophages reveal concentration-dependent increasing upon rhMCP-1 incubation. n = 3. (c) Upon rhMCP-1 (100 ng/mL) treatment and co-culture of macrophages-DPDO1 for 48 h, the mRNA expression of CD163 is increased, while the mRNA expression of CD80 is not significantly changed. (d) By rhMCP-1 incubation (100 ng/mL, 48 h) on M0-DPDO1 and M0-DPDO2, the LDH contents of supernatants are decreased. n = 3. (e) The diagram of co-culture of macrophages and PDOs. (f) Upon rhMCP-1 (100 ng/mL) incubation for 48 h, the numbers of cancer cells are increased. The upper lane is light images and the lower lane is images labeled by GFP. Scale bar = 30 μm (g) The images from Giemsa staining present decreased cellular disruption upon of rhMCP-1 incubation (100 ng/mL, 48 h) on DPDO1, compared to NC group. (h) The knockdown efficacy of CCR2 gene by Western blot. (i) The migrated cells stimulated by MCP-1/CCL2 was reversed by knockdown of CCR2. ^nsP > 0.05，*P < 0.05，***P < 0.001.

Fig. 6

The diagram of drug screening on PDOs of GC. The PDOs from DGC show drug-induced cellular senescence upon AURKi treatment. The senescent cancer cells produce MCP-1/CCL2 and attenuate innate immune response of macrophages in DGC.