Breast cancer cells can adapt to invading T lymphocytes by upregulating the immune checkpoint protein Programmed death ligand-1 (PD-L1) in response to increased local concentrations of interferon gamma (IFN-γ) and other cytokines. This allows a tumor to evade further immune targeting and reduces the innate immune response. Therefore, developing drugs to regulate checkpoint protein activity and expression is a popular approach in oncology today. It is also extremely important to develop in vitro tumor models for screening that are more predictive of drug responses in vivo. We describe here a culture system we developed to probe the effects immune and cancer cell interactions on the expression of a select panel of cytokines and immune checkpoint proteins. To determine if cytoarchitecture influences tumor cell responsiveness, cells were grown in both traditional monolayers and in complex 3D spheroid cultures in Ultra-Low Attachment (ULA) microplates. Human peripheral blood mononuclear cells (PBMCs), a heterogeneous population of blood cells, were cultured and stimulated with CD3/CD28 Dynabeads® to activate the T-cell population. PBMC activation induced the secretion of IFN-γ and other proteins which upregulated the expression of PD-L1 and other biomarkers in HCC38 (breast cancer-derived) cells. Using this model system, we discriminate between the effects of secreted factors from PBMCs on HCC38 cells compared to those caused by direct cellular contact in co-cultures. All biomarker expression data were measured using AlphaLISA no-wash homogeneous assays to assess protein concentrations in cellular supernatant and lysate fractions. Alpha technology requires very low sample volume (5 µL or less) allowing for technical replicates and parallel target testing from the same well of a culture plate and rapid screening of multiple biomarkers. Cellular health and proliferation were assessed using ATPlite luminescence assays and 3D imaging using the Opera PhenixTM High Content Screening System.