Characterization of esophageal squamous cell carcinoma cell lines sensitivity to Palbociclib

Background: Esophageal squamous cell carcinoma (eSCC) is a highly aggressive malignancy with poor prognosis and limited therapeutic options. Although immune checkpoint inhibitors such as nivolumab, have shown clinical benefit, particularly in patients with high PD-L1 expression, this subgroup represents only a small fraction of eSCC cases. CDK4/6 inhibitors such as palbociclib have only been tested as second-line agents in eSCC, often in combination with EGFR inhibitors, with minimal benefit. Methods: Our study evaluates palbociclib as a first-line therapy in treatment-naive eSCC models. Using a panel of 22 eSCC cell lines with integrated multi-omics and phenotypic assays. Results: We identified three response subtypes, resistant, delayed and arrested, correlated to Rb-pathway status. Interestingly, in delayed responders, palbociclib induced replication stress, DNA damage, and unprotected micronuclei enriched for cGAS, triggering activation of interferon-stimulated genes. Consistent with this, palbociclib enhanced immune cell infiltration in delayed eSCC spheroids within a preclinical vascularized 3D microfluidic system. Conclusions: Our study demonstrates that first-line palbociclib treatment unmasks intrinsic vulnerabilities in the CDK4/6-Rb axis and triggers innate immune activation in molecularly defined eSCC. Using a translationally relevant 3D vascularized microfluidic system, we provide evidence that early CDK4/6 inhibition not only stall cancer cell growth but also promotes immune cells recruitment. In conclusion, our study identifies palbociclib as a viable first-line therapeutic candidate in selected eSCC patients and uncover its immunomodulatory potential. These findings support further research into CDK4/6 inhibition as first-line treatment for eSCC and highlight its potential to influence the tumor immune microenvironment in ways that could improve responses to combination therapies. Overall design: We analyzed 22 human esophageal squamous cell carcinoma cell lines to investigate transcriptional responses to CDK4/6 inhibition. The cell lines used in this study were: TE-1, TE-4, TE-5, TE-6, TE-8, TE-9, TE-10, TE-11, TE-14, TE-15, KYSE-30, KYSE-70, KYSE-140, KYSE-150, KYSE-180, KYSE-270, KYSE-410, KYSE-450, KYSE-510, KYSE-520, COLO-680N, ECGI10 Each cell line was cultured under standard conditions and treated with either DMSO (vehicle control) or 1 µM palbociclib for 6 days. Total RNA was extracted at the end of treatment, and bulk RNA-seq was performed. For each condition, experiments were conducted in biological duplicate.