Tumor progression and metastasis are ultimately driven by a disruption in the organization and composition of the ECM with which resident cells interact. Cancer cell-ECM interactions have been shown to positively influence cancer cell survival and invasion by conferring adhesion-based resistance in response to chemotherapeutic drugs, and subsequently upregulating pathways driving metastasis into surrounding tissues. The frequent inability of chemotherapy to provide a long-term, complete cure may be attributed to this specific, adhesion-mediated resistance of malignant cells to chemotherapeutic interventions. Here, we present a platform designed to identify and perturb protective matrix properties at the nanoscale in a high-throughput manner, allowing for the conversion of chemoresistant cells into chemosensitive ones. Additionally, we examine the influence of nanoscale properties in combination with mechanical properties of the ECM, both in 2D and in 3D. Block copolymer micelle nanolithography (BCML) was utilized to create large, scalable arrays of specific ligands at defined nanoscale presentation of 50 and 75 nm spacing on glass substrates. Breast cancer cell lines plated on such arrays were then treated with chemotherapeutic drugs in order to identify the most protective ligand interactions, as determined by cell survival assays and immunofluorescence. Chemoresistance was found to greatly depend on ligand type and ligand presentation, e.g. laminin peptide is protective at 75 nm spacing but not 50 nm, while the opposite is true for RGD peptide. By utilizing BCML in conjunction with soft polymer transfer nanolithography, the influence of mechanical matrix properties in parallel with ligand presentation was analyzed on 2D substrates and inside 3D microchannels of 125 µm diameter. In both 2D and 3D systems, tumorigenic stiffness hydrogels (5 kPa) were more effective than softer, healthy breast tissue stiffness hydrogels (1 kPa) in upregulating cell area, polarity and motility, which have all been hypothesized to play a role in chemoresistance. Furthermore, mechanical properties were shown to influence chemosurvival in conjunction with ligand type and spacing, e.g. survival on laminin peptide was enhanced on 1 kPa vs. 5 kPa hydrogels for 50 nm spacing but not 75 nm. These scalable platforms enable the screening of several aspects of ECM-conferred chemoresistance in a highly defined manner, thereby allowing for new chemotherapeutic targets to be identified.