The role of stromal cells in resistance to cetuximab treatment in head and neck cancer

Introduction: Resistance to cetuximab, an antibody that blocks the epithelial growth factor receptor (EGFR), is a significant problem in the treatment of head and neck cancer (HNC) patients. Cetuximab is effective in a fraction of HNC patients, who eventually all develop resistance that leads to disease relapse. The resistance to cetuximab is known to be mediated, in part, by cells surrounding the tumor cells, stromal cells. However, the mechanisms underlying the migration, accumulation, and differentiation of these stromal cells are still unclear.

Material and method: We used HNC patient-derived xenografts (PDXs) models to investigate the interactions between tumor cells and their stroma. Five different PDXs were implanted in NOD.SCID mice and the response to cetuximab was measured. Molecular and pathological analyses of tumors using RNA sequencing (for three PDXs) and immunohistochemistry (IHC) (for all PDXs), respectively, were performed to gain insight on the changes that occur in the tumor and their stroma during cetuximab treatment. Specifically, staining of fibroblasts (aSMA), endothelial cells (CD31), tumor cells (KRT14), epithelial to mesenchymal markers (E-cadherin and Vimentin), and proliferation (KI67) were tested in PDXs treated with vehicle or cetuximab.

Results and discussion: All five PDXs responded to cetuximab indicated by a significant tumor growth inhibition. However, two of the five PDXs started to regrow and progress after a stable disease of 20 days, while in the other three, high sensitivity to cetuximab, we detected tumor shrinkage and even tumor elimination. RNA sequencing of 2 sensitive PDXs and 1 progressed PDX reveals that in the stromal compartment of the progressed PDX TGF beta signaling pathway was upregulated after cetuximab treatment, while in the sensitive PDXs, TGF beta pathway was downregulated. Furthermore, in the two sensitive PDXs, an upregulation of natural killer (NK) cell mediated cytotoxicity signature was detected. In the human compartment, the expression of several chemokines were altered after treatment, among them CXCL1 was the only chemokine that was highly expressed in the progressed tumor and its expression was altered in sensitive tumors after treatment with cetuximab. In addition, using IHC we observed a unique rearrangement of the cancer-associated fibroblast (CAFs) that surround the proliferating tumor cells after cetuximab treatment, while in the untreated tumors, CAFs were located between tumor cells. This phenomenon was highly prominent in the progressed PDXs.

Conclusion: Cetuximab treatment affects the plasticity and heterogeneity of tumor cells and their microenvironment (TME). Progression to cetuximab is associated with high levels of CXCL1 and upregulation of TGF beta signaling by the TME. In contrast, sensitivity to cetuximab is associated with low CXCL1 expression by tumor cells, downregulation of TGF-beta and upregulation of NK cells signatures.