IL-6 and IL-8, secreted by myofibroblasts in the tumor microenvironment, activate HES1 to expand the cancer stem cell population in early colorectal tumor.

Interaction between a tumor and its microenvironment is important for tumor initiation and progression. Cancer stem cells (CSCs) within the tumor interact with a microenvironmental niche that controls their maintenance and differentiation. We investigated the CSC-promoting effect of factors released from myofibroblasts into the microenvironment of early colorectal cancer tumors and its molecular mechanism. By messenger RNA microarray analysis, expression of HES1, a Notch signaling target, significantly increased in Caco-2 cells cocultured with 18Co cells (pericryptal myofibroblasts), compared to its expression in Caco-2 cells cultured alone. Caco-2 cells cultured in 18Co-conditioned media (CM) showed a significant increase in CD133+CD44+ cells and HES1 expression compared to that in Caco-2 cells cultured in regular media. Significant amounts of interleukin-6 (IL-6) and IL-8 were detected in 18Co-CM compared to levels in regular media. The 18Co-CM-induced increase in CD133+CD44+ cells was attenuated by IL-6- and IL-8-neutralizing antibodies. Furthermore, these neutralizing antibodies and inhibitors of STAT3 and gamma-secretase reduced the expression of HES1 induced in Caco-2 cells cultured in 18Co-CM. Immunohistochemical analysis of human tissues revealed that IL-6, IL-8, and HES1 expression increased from normal to adenoma, and from adenoma to cancer tissues. In addition, IL-6 and HES1 expression was positively correlated in early colorectal cancer tissues. In conclusion, the increase of CSCs by myofibroblasts could be mediated by IL-6/IL-8-induced HES1 activation in the tumor microenvironment. Based on these data, the IL-6/IL-8-mediated Notch/HES1 and STAT3 pathway, through which CSCs interact with their microenvironment, might be a potential target for the prevention and treatment of colorectal tumors.

Kim B ，Seo Y ，Kwon JH ，Shin Y ，Kim S ，Park SJ ，Park JJ ，Cheon JH ，Kim WH ，Il Kim T ... -  《-》

The role of myofibroblasts in upregulation of S100A8 and S100A9 and the differentiation of myeloid cells in the colorectal cancer microenvironment.

S100A8/A9 and myeloid cells in the tumor microenvironment play an important role in cancer invasion and progression, and the effect of tumor-infiltrated myofibroblasts on myeloid cells in the tumor microenvironment is relatively unknown. Accordingly, we investigated the role of myofibroblasts in the upregulation of S100A8/A9 as well as in the differentiation of myeloid cells in the colorectal cancer (CRC) microenvironment. To investigate the interactions among cancer cells, myofibroblasts, and inflammatory cells in the microenvironment of CRC, we used 10 CRC cell lines, 18CO cells and THP-1 cells, which were co-cultured with each other or cultured in conditioned media (CM) of other cells. Expression of S100A8/A9 was evaluated via Western blot, immunohistochemical staining and immunofluorescence. The secreted factors from the cell lines were analyzed using cytokine antibody array. Flow cytometry analysis was performed to analyze the differentiation markers of myeloid cells. 18CO CM induced increased expression of S100A8/A9 in THP-1 cells. Increased expression of S100A8/A9 was noted in inflammatory cells of the peri- and intra-tumoral areas, along with myofibroblasts in colon cancer tissue. S100A8/A9-expressing inflammatory cells also exhibited CD68 expression in colon cancer tissue, and 18CO CM induced differentiation of THP-1 cells into myeloid-derived suppressor cells (MDSCs) or M2 macrophages expressing S100A8/A9. Significant amounts of IL-6 and IL-8 were detected in 18CO CM, compared to those in both controls and THP-1 CM, and tumor-infiltrated myofibroblasts expressed IL-8 in colon cancer tissue. Finally, neutralizing antibodies to IL-6 and IL-8 attenuated 18CO CM-induced increased expression of S100A8/A9. The upregulation of S100A8/A9 in tumor-infiltrated myeloid cells could be triggered by IL-6 and IL-8 released from myofibroblasts, and myofibroblasts might induce the differentiation of myeloid cells into S100A8/9-expressing MDSCs or M2 macrophages in the CRC microenvironment.

Kim JH ，Oh SH ，Kim EJ ，Park SJ ，Hong SP ，Cheon JH ，Kim TI ，Kim WH ... -  《-》

Nonsteroidal anti-inflammatory drugs suppress cancer stem cells via inhibiting PTGS2 (cyclooxygenase 2) and NOTCH/HES1 and activating PPARG in colorectal cancer.

Cancer stem cells (CSCs) play a pivotal role in cancer relapse or metastasis. We investigated the CSC-suppressing effect of nonsteroidal anti-inflammatory drugs (NSAIDs) and the relevant mechanisms in colorectal cancer. We measured the effect of NSAIDs on CSC populations in Caco-2 or SW620 cells using colosphere formation and flow cytometric analysis of PROM1 (CD133)(+) CD44(+) cells after indomethacin treatment with/without prostaglandin E2 (PGE2) or peroxisome proliferator-activated receptor γ (PPARG) antagonist, and examined the effect of indomethacin on transcriptional activity and protein expression of NOTCH/HES1 and PPARG. These effects of indomethacin were also evaluated in a xenograft mouse model. NSAIDs (indomethacin, sulindac and aspirin), celecoxib, γ-secretase inhibitor and PPARG agonist significantly decreased the number of colospheres formation compared to controls. In Caco-2 and SW620 cells, compared to controls, PROM1 (CD133)(+) CD44(+) cells were significantly decreased by indomethacin treatment, and increased by 5-fluorouracil (5-FU) treatment. This 5-FU-induced increase of PROM1 (CD133)(+) CD44(+) cells was significantly attenuated by combination with indomethacin. This CSC-inhibitory effect of indomethacin was reversed by addition of PGE2 and PPARG antagonist. Indomethacin significantly decreased CBFRE and increased PPRE transcriptional activity and their relative protein expressions. In xenograft mouse experiments using 5-FU-resistant SW620 cells, the 5-FU treatment combined with indomethacin significantly reduced tumor growth, compared to 5-FU alone. In addition, treatment of indomethacin alone or combination of 5-FU and indomethacin decreased the expressions of PROM1 (CD133), CD44, PTGS2 (cyclooxygenase 2) and HES1, and increased PPARG expression. NSAIDs could selectively reduce the colon CSCs and suppress 5-FU-induced increase of CSCs via inhibiting PTGS2 (cyclooxygenase 2) and NOTCH/HES1, and activating PPARG.

Moon CM ，Kwon JH ，Kim JS ，Oh SH ，Jin Lee K ，Park JJ ，Pil Hong S ，Cheon JH ，Kim TI ，Kim WH ... -  《-》

The effectiveness of an anti-human IL-6 receptor monoclonal antibody combined with chemotherapy to target colon cancer stem-like cells.

Ying J ，Tsujii M ，Kondo J ，Hayashi Y ，Kato M ，Akasaka T ，Inoue T ，Shiraishi E ，Inoue T ，Hiyama S ，Tsujii Y ，Maekawa A ，Kawai S ，Fujinaga T ，Araki M ，Shinzaki S ，Watabe K ，Nishida T ，Iijima H ，Takehara T ... -  《-》

Co-culture of ovarian cancer stem-like cells with macrophages induced SKOV3 cells stemness via IL-8/STAT3 signaling.

Among recent concepts in the cancer biology field, the tumor microenvironment is highly associated with cancer stem cells, and plays a key role in tumor progression. This study aimed to explore the mechanism that the stemness induction of SKOV3 cell line by macrophages derived from THP-1 cells, which was co-cultured with SKOV3-derived ovarian cancer stem-like cells (OCSLCs). Sphere formation, soft agar colony formation, and expression levels of CD133 and CD44 were assessed to reflect OCSLC properties. ELISA was used to evaluate secretion profile changes in macrophages co-cultured with or without SKOV3-derived OCSLCs. For mechanistic evaluation, rhIL-8, IL-8 neutralizing antibody (IL-8 Ab), signal transducer and activator of transcription 3 (STAT3) shRNA and STAT3 cDNA were used. The results showed that IL-10, VEGF, MMP-9, IL-8 secretion and CD163 and STAT3 expression levels in macrophages co-cultured with OCSLCs were increased compared with those from THP-1 cells, while IL-12 and NO amounts were significantly reduced, reflecting M2 macrophage polarization. Addition of rhIL-8 to THP-1 cell conditioned media promoted M2 macrophage polarization and stemness in SKOV3 cells, which were suppressed by IL-8 Ab addition to co-culture conditioned media. Consistently, overexpression of STAT3 induced M2 macrophage polarization and stemness in SKOV3 cells, which were inhibited by STAT3 knockdown in macrophages from THP-1 cells. Importantly, STAT3 overexpression rescued the effects of IL-8 Ab on M2 macrophage polarization and stemness in SKOV3 cells. These results suggested that stemness induction in SKOV3 cells by macrophages co-cultured with SKOV3-derived OCSLCs involved IL-8/STAT3 signaling.

Ning Y ，Cui Y ，Li X ，Cao X ，Chen A ，Xu C ，Cao J ，Luo X ... -  《-》