<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

Clinical and genomic profiles of 10 CTOS lines.

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

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

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

In vivo cetuximab sensitivity assay revealed diverse effect on 10 CTOS-derived xenogfrafts.

<p>A, Growth curves of subcutaneous tumors originating from colorectal CTOS lines. Blue, treated with vehicle; orange, cetuximab (20 mg/kg); green, cetuximab (60 mg/kg). Mean±SD is shown. N = 4–6 in each treated group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; 60 mg/kg cetuximab versus control; two-way ANOVA with Bonferroni post-test. Regression, partially responsive, and resistant are explained in the text. The type of <i>KRAS</i> mutant is indicated in superscript to the left of the line name. B, Microscopic images of vehicle-treated xenografts in A stained with EGFR antibody. Scale bar, 50 μm. Grading by staining intensity is shown. C, Waterfall plot of cetuximab-treated tumor growth (differences from baseline). The average sizes of cetuximab (60 mg/kg) treated tumors at day 21 were subtracted by the sizes at day 0 and the ratio compared to the average sizes at day 0 as follows: (V_Cmab (day 21)—V_Cmab (day 0))/ V_Cmab (day 0) x 100. Red bars, wildtype <i>KRAS</i> tumors; blue, <i>KRAS</i> mutants. D, Growth reduction from control (vehicle treated) tumors. The average sizes of vehicle-treated tumors at day 11 were subtracted by the sizes of tumors treated with cetuximab (60 mg/kg) and the ratio compared to the average sizes at day 11 as follows: (V_vehicle(day 11)- V_Cmab(day 11))/ V_vehicle(day 11) x 100.</p

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

<p>A, Screening of 71 drugs using reconstituted C45 spheroids. The spheroids were cultured with 100 nM of drug for 7 days. The drugs for which the relative ATP content was less than 0.5 are indicated. Mean values are shown. N = 4. B, Dose-response curve of trametinib in four CTOS lines (the partially responsive group, C45 and CB3; the resistant group, C75 and C48). The type of <i>KRAS</i> mutant is indicated in superscript to the left of the line name. C, Lysates of reconstituted spheroids from four CTOS lines were subjected to Western blotting. The spheroids were treated with the indicated doses of trametinib for 4 h. The antibodies used are indicated. D, Relative ATP content of reconstituted spheroids cultured for 7 days with cetuximab alone at the indicated doses (black bars), or in combination with 10 nM of trametinib (gray bars). Mean±SD is shown. N = 6. Significance of the decrease in relative ATP content. *P < 0.05, **P < 0.01, ****P < 0.0001, versus 0; one-way ANOVA with Bonferroni post-test. E, Western blots of lysates from the reconstituted spheroids treated with or without 100 nM cetuximab, 5 nM trametinib, or a combination of 100 nM cetuximab and 5 nM trametinib for 2 h with or without stimulation with 10 ng/ml EGF for 15 min.</p

Establishment of ex vivo cetuximab sensitivity assays.

<p>A, Growth of C132 CTOSs cultured with the indicated doses of cetuximab for 7 days. The relative growth at day 7 adjusted by day 0 is shown. Culture media are indicated. Mean±SD is shown. N = 3–6. Significance of the decrease in relative growth. *P < 0.05, versus 0; one-way ANOVA with Bonferroni post-test. B, Western blots of lysates from C132 CTOSs cultured in the indicated media for 24 h. GF-, basal medium without any growth factor; SM, the standard CTOS medium; FBS, the FBS medium. Each antibody is indicated. C, Western blots of lysates from C132 CTOSs treated with or without cetuximab (100 nM) for 2 h. CTOSs were cultured in the indicated media for 24 h before cetuximab treatment. Cmab, cetuximab. D, Growth of C132 CTOSs cultured with the indicated doses of cetuximab for 7 days. CTOSs were cultured in the FBS medium with (gray bars) or without (black bars, same as Fig 2A) 10 ng/ml HRG. The relative growth at day 7 adjusted by day 0 is shown. Mean±SD is shown. N = 5–6. Significance of the decrease in relative growth. *P < 0.05, versus 0; one-way ANOVA with Bonferroni post-test. E, Results of the reconstituted spheroid-based assay. The viable cell number in the spheroids was evaluated by ATP content, and the ratio to that of non-treated spheroids is shown. CTOSs were cultured for 7 days in the indicated media with the indicated doses of cetuximab. Mean±SD is shown. N = 5–6. Significance of the decrease in relative ATP content. ****P < 0.0001, versus 0; one-way ANOVA with Bonferroni post-test. F, Western blot evaluation of intracellular signaling ex vivo. Reconstituted spheroids generated from C132 CTOSs were treated with or without 100 nM cetuximab for 2 h and then stimulated with or without 10 ng/ml of EGF for 15 min.</p

Representative PET images of mice bearing CTOS C45 xenografts.

<p>Serial PET images of mice bearing C45 xenograft (white arrow) injected with [⁸⁹Zr]Mab#58 (upper panels) or similarly ⁸⁹Zr-labeled control IgG.</p

Representative PET images of mice bearing both HER3 overexpressing HER3/RH7777 and parent RH7777 xenografts.

<p>Serial PET images (maximum-intensity-projection (MIP)) of a mouse bearing HER3/RH7777 (right thigh, white arrow) and RH7777 (left thigh, orange arrow) tumors at days 1, 2, 4, and 6 after an intravenous injection of 3.7 MBq [⁸⁹Zr]Mab#58.</p

Representative immunohistochemical staining of HER3/RH7777 (A) and RH7777 (B) xenografts for HER3 expression.

<p>Representative immunohistochemical staining of HER3/RH7777 (A) and RH7777 (B) xenografts for HER3 expression.</p