proox c21  (BioSpherix)

Journal: Cancer cell

Article Title: Cholinergic signaling via muscarinic receptor 3 promotes colorectal cancer progression directly and indirectly through stroma-derived Netrin-1

doi: 10.1016/j.ccell.2026.06.018

Figure Lengend Snippet: (A) Experimental scheme showing rectal AKP transplantation into Lepr-Cre(+);Chrm3 fl/fl and control mice. IVIS, in vivo imaging. (B and C) Mouse survival (B) and tumor-derived luciferase signals (C). n=6 ( Lepr-Cre(−);Chrm3 fl/fl ) and 9 mice ( Lepr-Cre(+);Chrm3 fl/fl ). (D) Non-phosphorylated YAP/PDGFRA co-IF. Red arrowheads; non-phosphorylated (active) YAP in PDGFRA + CAFs. cKO, conditional KO. n=5 mice. (E) NTN1/PDGFRA co-IF. n=5 mice. (F) VAChT IHC. Red arrowheads; VAChT + nerves. n=5 mice. (G) Experimental scheme showing rectal AKP transplantation with daily intratumor injection of the hM3Dq agonist C21 (0.03 mg/kg) from Day 8. (H and I) Mouse survival (H) and tumor-derived luciferase signals (I). n=8 ( Lepr-Cre;LSL-hM3Dq(−);Ntn1 WT ), 7 ( Lepr-Cre;LSL-hM3Dq(+);Ntn1 WT ), 7 ( Lepr-Cre;LSL-hM3Dq(−);Ntn1 fl/fl ), and 7 mice ( Lepr-Cre;LSL-hM3Dq(+);Ntn1 fl/fl ). (J) Non-phosphorylated YAP/PDGFRA co-IF. Red/purple arrowheads; non-phosphorylated YAP in PDGFRA + CAFs in the Lepr-Cre;LSL-hM3Dq(+);Ntn1 WT / Lepr-Cre;LSL-hM3Dq(+);Ntn1 fl/fl groups. n=4 mice. (K) NTN1/PDGFRA co-IF. n=4 mice. (L) VAChT IHC. Green/red arrowheads:VAChT + nerves in the Lepr-Cre;LSL-hM3Dq(−);Ntn1 WT / Lepr-Cre;LSL-hM3Dq(+);Ntn1 WT groups. n=4 mice. Scale bars, 100 μm (A and G), 50/5 μm (main/insets; D, E, J, and K), 50 μm (F and L). Log-rank test (B and H), two-way repeated-measures ANOVA with post-hoc Sidak’s multiple comparison test at week 2 (C and I), two-tailed unpaired Student’s t-test (D-F), and two-way ANOVA followed by Tukey’s post-hoc multiple comparison tests (J-L). Mean±s.e.m. (C-F and I-L). See also Figures S5 .

Article Snippet: A Hamilton syringe holding a 33-gauge needle was used to inject C21 (0.03 mg/kg; HY-100234A, MedChemExpress; a volume of 10 μL, dissolved in DPBS) into the rectal tumor percutaneously through the anal skin.

Techniques: Expressing, Transplantation Assay, Control, In Vivo Imaging, Derivative Assay, Luciferase, Injection, Comparison, Two Tailed Test, Immunohistochemistry

Journal: Cancer cell

Article Title: Cholinergic signaling via muscarinic receptor 3 promotes colorectal cancer progression directly and indirectly through stroma-derived Netrin-1

doi: 10.1016/j.ccell.2026.06.018

Figure Lengend Snippet: (A) Luciferase (luc)-expressing AKP tumor organoids were co-treated with ACh and an AChR antagonist for 72 h. AKP tumor-derived luc signals were measured as a surrogate for viable cell numbers. Drug concentration as in Figure 2A . n=4. (B) Luc signals from control gRNA/g Chrm3 -expressing luc + AKP organoids treated (72 h). Bethanechol (Beth), 10 μM. n=4. (C) Gene set enrichment analysis of the YAP gene signature 53 using bulk RNA-seq from Beth-/vehicle-treated AKP organoids. The normalized enrichment score (NES) and false discovery rate (FDR)-adjusted P value are shown. n=3. (D) Western blotting of control gRNA/g Chrm3 -expressing AKP tumor cells treated (20 min). Red dotted rectangles: dephosphorylation of LATS1/YAP by ACh/Beth treatment. FR900359 (FR, Gq inhibitor), 1 μM. (E) Luc signals from luc + AKP organoids treated (72 h). VP, verteporfin (YAP inhibitor, 0.5 μM). n=5. (F) Experimental scheme showing rectal AKP transplantation with daily intratumor injection of the hM3Dq agonist C21 (0.03 mg/kg) from Day 8. (G and H) Mouse survival (G) and tumor-derived luc signals (H). n=10 ( Chat-Cre(−);hM3Dq mice; control AKP), 9 ( Chat-Cre(+);hM3Dq mice; control AKP), 9 ( Chat-Cre(−);hM3Dq mice; g Chrm3 AKP), and 9 mice ( Chat-Cre(+);hM3Dq mice; g Chrm3 AKP). (I) Non-phosphorylated YAP/luc (a marker for luc + AKP cancer cells) co-IF. Red arrowheads; non-phosphorylated YAP in luc + cancer cells. n=5 mice. Scale bars, 100 μm (A and F) and 50/5 μm (main/insets; I). One-way ANOVA followed by Tukey’s post-hoc multiple comparison tests (A, B, and E), gene set permutation tests (C), log-rank test (G), two-way repeated-measures ANOVA with post-hoc Sidak’s multiple comparison test at week 2 (H), and two-way ANOVA followed by Tukey’s post-hoc multiple comparison tests (I). Mean±s.e.m. (A, B, E, H, and I). See also Figures S6 and S7 ; Tables S3 and S4 .

Article Snippet: A Hamilton syringe holding a 33-gauge needle was used to inject C21 (0.03 mg/kg; HY-100234A, MedChemExpress; a volume of 10 μL, dissolved in DPBS) into the rectal tumor percutaneously through the anal skin.

Techniques: Activation Assay, Luciferase, Expressing, Organoids, Derivative Assay, Concentration Assay, Control, Analysis, RNA Sequencing, Western Blot, De-Phosphorylation Assay, Transplantation Assay, Injection, Marker, Comparison