Fatty Acid Oxidation Supports Lymph Node Metastasis of Cervical Cancer via Acetyl‐CoA‐Mediated Stemness

Abstract Accumulating evidence indicates that metabolic reprogramming of cancer cells supports the energy and metabolic demands during tumor metastasis. However, the metabolic alterations underlying lymph node metastasis (LNM) of cervical cancer (CCa) have not been well recognized. In the present study, it is found that lymphatic metastatic CCa cells have reduced dependency on glucose and glycolysis but increased fatty acid oxidation (FAO). Inhibition of carnitine palmitoyl transferase 1A (CPT1A) significantly compromises palmitate‐induced cell stemness. Mechanistically, FAO‐derived acetyl‐CoA enhances H3K27 acetylation (H3K27Ac) modification level in the promoter of stemness genes, increasing stemness and nodal metastasis in the lipid‐rich nodal environment. Genetic and pharmacological loss of CPT1A function markedly suppresses the metastatic colonization of CCa cells in tumor‐draining lymph nodes. Together, these findings propose an effective method of cancer therapy by targeting FAO in patients with CCa and lymph node metastasis.


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Figure S1 Metastatic cervical cancer cells in lymph node displayed increased fatty acid oxidation.A. Differentially expressed transcripts of HeLa LNM2 cells compared with HeLa PR cells involved in cell metabolism were depicted in a volcano plot.The black dotted lines indicated a fold-change threshold of 2, and the P value cutoff (P = 0.05).B, C. Gene set enrichment analyses of pathways in Figure S1A.The fatty acid metabolism pathway was significantly activated while glycolysis-gluconeogenesis pathway showed an opposite effect.

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Figure S2 Inhibition of CPT1A had no significant effect on primary tumor growth.A. Representative images of immunofluorescence staining of Ki67 and Cleaved-caspase3 after footpad injection of HeLa LNM2 cells under different treatments (n = 6).B. Representative images of immunofluorescence staining of Ki67 and Cleaved-caspase3 after footpad injection of SiHa LNM2 cells under different treatments (n = 6).Error bars represent the means ± SD.Statistical analyses were performed using a two-tailed Student's t-test.ns, no significance.

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Figure S3 High-fat diet promoted lymph node metastasis of CCa.A, B. Representative images and the LNs volume in different groups (n = 6).C, D. Representative images of immunohistochemistry staining of pan-cytokeratin and metastatic area in popliteal LNs.Error bars represent the means ± SD.Statistical analyses were performed using a two-tailed Student's t-test.***P < 0.001; ****P < 0.0001.

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Figure S4 Depletion of macrophages had no significant influence on LNM of CCa. A. Representative Immunofluorescence micrographs of F4/80+ macrophage cells in vehicle-and clodronate-treated CCa tumors.Bar = 50 μm.B. Quantification of F4/80+ signals.C. Representative HE and immunohistochemical micrographs of lymph nodes in vehicle-and clodronate-treated groups (n = 6).D. Metastatic area of lymph nodes in each group (n = 6).Error bars represent the means ± SD.Statistical analyses were performed using a two-tailed Student's t-test.***P < 0.001; ****P < 0.0001, ns, no significance.

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Figure S5 Inhibition of FAO had no significant influence on macrophage infiltration and polarization.A, B. Representative immunofluorescent micrographs of total and M2-like macrophages in primary tumor and lymph node under different conditions.Bar = 100μm, and quantification of F4/80 + and CD206 + F4/80 + signals in the left images.Error bars represent the

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Figure S6 LN-metastatic cervical cancer cells displayed increased stemness.A. Sphere formation assay of HeLa LNM2, SiHa LNM2, HeLa PR, and SiHa PR cells.B, C. The mRNA expression levels of stemness-related genes in CCa cells, Bar = 150μm.D. Relative protein levels of stemness-related genes in CCa cells.E, F. Percentage of ALDH1 + cells detected by flow cytometry.Numerical values are presented as percentage.Each experiment was performed at least three times independently.Error bars represent the means ± SD.Statistical analyses were performed using a two-tailed Student's t-test.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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Figure S7 Fatty acid oxidation participated in the regulation of CCa cells stemness.A, B. Percentage of ALDH1 + cells detected by flow cytometry in the indicated groups.Numerical values are presented as percentage.C-E.Percentage of ALDH1 + cells detected by flow cytometry in the indicated groups of HeLa LNM2 and SiHa LNM2 cells.Numerical values are presented as percentage.Each experiment was performed at least three times independently.Error bars represent the means ± SD.Statistical analyses were performed using a two-tailed Student's t-test.****P < 0.0001.

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Figure S8 Fatty acid oxidation derived acetyl-CoA promoted H3K27 acetylation and CCa cells stemness.A. The expression levels of total acetyl-H3 and H3K27ac were analyzed by Western blotting in CCa cells treated with BSA or PA.B. The expression levels of total acetyl-H3 and H3K27Ac were analyzed by western blotting in the indicated groups.C. A dose-dependent effect accompanied with acetate concentrations.D, E. Sphere formation and sphere formation rate of CCa cells under different dose of acetate supplement.Bar = 150μm.F. Percentage of ALDH1 + cells detected by flow cytometry in CCa cells under different dose of acetate supplement.

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Figure S9 CPT1A was highly expressed in metastatic CCa cells.A. Results of multiple immunofluorescence showed the co-location of CPT1A, CK, and CD44.