Downregulation of AKT phosphorylation is necessary for induction of dormant status in primary colorectal cancer.

<p>A) CTOS growth was measured by size relative to day 0. C45 CTOS samples were cultured in medium with (GF+) or without (GF−) growth factors. B) Representative images of C45 CTOS cultured in indicated conditions. Scale bar  = 100 µm. C) Regrowth of CTOS in dormant status after re-oxygenation and exposure to growth factor–containing medium. D) Immunohistochemistry of C45 CTOS cultured in indicated conditions for 1 day. TUNEL staining was at day 14. Scale bar  = 50 µm. E) Immunoblot of AKT/mTORC1 signaling and HIF-1α in C45 CTOS cultured in indicated conditions.</p

Downregulation of AKT phosphorylation is important for induction of dormant status.

<p>A) Immunoblot of the AKT/mTORC1 or ERK/p38 MAPK pathway in AsPC-1 cells cultured in hypoxia. B) Immunoblot of AKT signaling and HIF-1α in AsPC-1 cells expressing vector control, AKT-wt, or AKT-3A (inactive). C) Cell cycle status of the cells at day 7 in hypoxia. Percentages of the cells in S phase are indicated above the plot and in the right graph. D) ATP turnover measured by adding inhibitor cocktail for glycolysis and oxidative phosphorylation. N1, normoxia 1 day; H1, hypoxia 1 day; H7, hypoxia 7 days. E, F) Viable cell number (E) and percent cell death (F) of AsPC-1 cells cultured in hypoxia. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

AsPC-1 cells can be in a dormant status in chronic hypoxia.

<p>Viable cell number (A) and percent cell death (B) of AsPC-1 cells cultured in normoxia (20% O₂) or hypoxia (1% O₂). C) Phase-contrast and PI-stained images of AsPC-1 cells cultured under the indicated conditions. Scale bar  = 50 µm. D) Cell cycle analysis of AsPC-1 cells at day 1 in normoxia or day 1 and day 7 in hypoxia. Cells were pulsed with BrdU for 2 h and analyzed by flow cytometry after staining with anti-BrdU antibody and PI. Percentages of the cells in S phase are indicated. E) Re-growth of AsPC-1 cells in a dormant status. AsPC-1 cells were cultured in hypoxia for 14 days, and the cell counts were monitored after re-seeding in normoxic conditions.</p

Metabolic processes are suppressed under chronic hypoxia.

<p>A) Lactate production rate (left) was calculated from lactate concentration and integral cell number at indicated periods. O₂ consumption rate (middle) was measured using a Clark type oxygen electrode. ATP turnover (right) was calculated from the lactate production rate and O₂ consumption rate (dark gray: lactate; light gray: oxygen). N1, normoxia 1 day; H1, hypoxia 1 day; H7, hypoxia 7 days. ** <i>p</i><0.01, ***<i>p</i><0.001. B) Quantitative RT-PCR of a glucose transporter and glycolytic enzymes in AsPC-1 cells cultured in hypoxia for the indicated days.</p

Dormancy of Cancer Cells with Suppression of AKT Activity Contributes to Survival in Chronic Hypoxia

<div><p>A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, that is exceptionally able to survive for weeks under 1% oxygen conditions while most tested cancer cell lines die after only some days under these conditions. In chronic hypoxia, AsPC-1 cells entered a state of dormancy characterized by no proliferation, no death, and metabolic suppression. They reversibly switched to active status after being placed again in optimal culture conditions. ATP turnover, an indicator of energy demand, was markedly decreased and accompanied by reduced AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death <i>in vitro</i> and a decreased number of dormant cells <i>in vivo</i>. In contrast to most cancer cell lines, primary-cultured colorectal cancer cells easily entered the dormant status with AKT suppression under hypoxia combined with growth factor–depleted conditions. Primary colorectal cancer cells in dormancy were resistant to chemotherapy. Thus, the ability to survive in a deteriorated microenvironment by entering into dormancy under chronic hypoxia might be a common property among cancer cells. Targeting the regulatory mechanism inducing this dormant status could provide a new strategy for treating cancer.</p></div

Primary colorectal cancer in dormant status is resistant to chemotherapy.

<p>A) CTOS samples were cultured in medium with (GF+) or without (GF−) growth factors, and under 20% O₂ or 1% O₂ conditions. 5FU or SN38 were added to medium and treated for 7 days (indicated by black bars). At day 7, medium was changed to fresh StemPro hESC containing growth factors (black arrows), and CTOS samples were allowed to regrow under 20% O₂. B) Representative images of CTOS samples in (A). Scale bar  =  100 µm.</p

<i>In vivo</i> and <i>ex vivo</i> cetuximab sensitivity assay using three-dimensional primary culture system to stratify <i>KRAS</i> mutant colorectal cancer

<div><p>In clinic, cetuximab, an anti-EGFR antibody, improves treatment outcomes in colorectal cancer (CRC). <i>KRAS</i>-mutant CRC is generally resistant to cetuximab, although difference of the sensitivity among <i>KRAS</i>-mutants has not been studied in detail. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method for cancer cells. We applied CTOS method to investigate whether ex vivo cetuximab sensitivity assays reflect the difference in sensitivity in the xenografts. Firstly, in vivo cetuximab treatment was performed with xenografts derived from 10 CTOS lines (3 <i>KRAS</i>-wildtype and 7 <i>KRA</i>S mutants). All two CTOS lines which exhibited tumor regression were <i>KRAS</i>-wildtype, meanwhile all <i>KRAS</i>-mutant CTOS lines grew more than the initial size: were resistant to cetuximab according to the clinical evaluation criteria, although the sensitivity was quite diverse. We divided <i>KRAS</i>-mutants into two groups; partially responsive group in which cetuximab had a substantial growth inhibitory effect, and resistant group which exhibited no effect. The ex vivo signaling assay with EGF stimulation revealed that the partially responsive group, but not the resistant group, exhibited suppressed ERK phosphorylation ex vivo. Furthermore, two lines from the partially responsive group, but none of the lines in the resistant group, exhibited a combinatory effect of cetuximab and trametinib, a MEK inhibitor, ex vivo and in vivo. Taken together, the results indicate that ex vivo signaling assay reflects the difference in sensitivity in vivo and stratifies KRAS mutant CTOS lines by sensitivity. Therefore, coupling the in vivo and ex vivo assays with CTOS can be a useful platform for understanding the mechanism of diversity in drug sensitivity.</p></div

Response of ERK phosphorylation to cetuximab treatment stratified <i>KRAS</i> mutants into two groups ex vivo.

<p>The result of the reconstituted spheroid-based assay in each CTOS line is shown. The CTOS lines are ordered according to the results of the in vivo assay and the grouping indicated below. The type of <i>KRAS</i> mutant is indicated in superscript to the left of the line name. Upper panels: relative ATP content of reconstituted spheroids cultured with the indicated doses of cetuximab for 7 days. Mean±SD is shown. N = 3–6. Significance of the decrease in relative ATP content. *P < 0.05, ***P < 0.001, ****P < 0.0001, versus 0; one-way ANOVA with Bonferroni post-test. Lower panels: ex vivo signaling assay by Western blot. Reconstituted spheroids were treated with or without 100 nM cetuximab for 2 h and then stimulated with or without 10 ng/ml EGF for 15 min. Cmab, cetuximab.</p

Clinical and genomic profiles of 10 CTOS lines.

<p>Clinical and genomic profiles of 10 CTOS lines.</p

Combination of cetuximab and trametinib was more effective in the partially responsive group in vivo.

<p>Growth curves of subcutaneous tumors generated by four CTOS lines (the partially responsive group, C45 and CB3; the resistant group, C75 and C48). Blue, treated with vehicle; orange, cetuximab (20 mg/kg) alone; green, trametinib (0.3 mg/kg) alone; purple, combination of cetuximab (20 mg/kg) and trametinib (0.3 mg/kg). Mean±SD is shown. N = 6 in each treated group. *P < 0.05, **P < 0.01, mono therapy with trametinib versus combination; two-way ANOVA with Bonferroni post-test. The type of <i>KRAS</i> mutant is indicated in superscript to the left of the line name.</p