HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography–mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.

The COVID-19 pandemic highlighted testing inequities in developing countries. Lack of lateral flow test (LFT) manufacturing capacity was a major COVID-19 response bottleneck in low- and middle-income regions. Here we report the development of an open-access LFT for SARS-CoV-2 detection comparable to commercial tests that requires only locally available supplies. The main critical resource is a locally developed horse polyclonal antibody (pAb) whose sensitivity and selectivity are greatly enhanced by affinity purification. We demonstrate that these Abs can perform similarly to commercial monoclonal antibodies (mAbs), as well as mAbs and other pAbs developed against the same antigen. We report a workflow for test optimization using nasopharyngeal swabs collected for RT-qPCR, spiked with the inactivated virus to determine analytical performance characteristics as the limit of detection, among others. Our final prototype showed a performance similar to available tests (sensitivity of 83.3% compared to RT-qPCR, and 90.9% compared to commercial antigen tests). Finally, we discuss the possibility and the challenges of utilizing affinity-purified pAbs as an alternative for the local development of antigen tests in an outbreak context and as a tool to address inequalities in access to rapid tests.

Resistance to targeted therapy in HER2-positive breast cancer remains a clinical challenge, especially for patients with relapsed or metastatic disease. Particularly, persistent activation of hypoxia-inducible factor 1 (HIF-1) signalling is well documented in the context of trastuzumab and trastuzumab emtansine resistance. To achieve a deeper understanding of how HIF-1 activity modulates the response to anti-HER2 treatment, we functionally characterized a cellular model of hypoxia-induced drug resistance for HER2-positive breast cancer using shotgun proteomics. By global phosphoproteomics profiling, the Rac1 pathway was identified as one of the most enriched signalling networks under hypoxia. Furthermore, the selective Rac1 blockade with the 1A-116 small-molecule inhibitor sensitised HER2-positive cells to trastuzumab in both 2D and 3D culture systems. Altogether, our findings demonstrate that hypoxic conditions induce the resistance of HER2-positive breast cancer cells to targeted therapy and suggest the therapeutic potential of Rac1 inhibition to enhance trastuzumab efficacy.