Simple SummaryLung cancer is one of the most common cancers in the world, and despite improvements in treatment, it remains a significant health burden. B7-H3 is a member of the B7 immune checkpoint family of membrane proteins and shows increased expression in lung cancers; however, the functional consequences of this to cancer cell signalling remain unclear. Here, we show that B7-H3 forms a complex with the metabolic enzyme IMPDH2, and this protects cancer cells from oxidative stress and resulting apoptosis triggered by chemotherapy. The removal of B7-H3 from cancer cells in 2D results in sensitisation to chemotherapeutic stress and reduces cell viability in an IMPDH2-dependent manner. However, the loss of B7-H3 in 3D spheroid models results in enhanced IMPDH2-dependent spheroid growth, likely due to the weakening of cell–cell adhesion. These findings demonstrate that B7-H3 functionally contributes to metabolic signalling in lung cancer cells in the absence of immune cell interactions.AbstractLung cancer is one of the most common cancers worldwide, and despite improvements in treatment regimens, patient prognosis remains poor. Lung adenocarcinomas develop from the lung epithelia and understanding how specific genetic and environmental factors lead to oncogenic transformation in these cells is of great importance to define the pathways that contribute to tumorigenesis. The recent rise in the use of immunotherapy to treat different cancers has prompted the exploration of immune modulators in tumour cells that may provide new targets to manipulate this process. Of these, the B7 family of cell surface receptors, which includes PD-1, is of particular interest due to its role in modulating immune cell responses within the tumour microenvironment. B7-H3 (CD276) is one family member that is upregulated in many cancer types and suggested to contribute to tumour–immune interactions. However, the function and ligand(s) for this receptor in normal lung epithelia and the mechanisms through which the overexpression of B7-H3 regulate cancer progression in the absence of immune cell interactions remain unclear. Here, we present evidence that B7-H3 is associated with one of the key rate-limiting metabolic enzymes IMPDH2, and the localisation of this complex is altered in human lung cancer cells that express high levels of B7-H3. Mechanistically, the IMPDH2:B7-H3 complex provides a protective role in cancer cells to escape oxidative stress triggered by chemotherapy, thus leading to cell survival. We further demonstrate that the loss of B7-H3 in cancer cells has no effect on growth or migration in 2D but promotes the expansion of 3D spheroids in an IMPDH2-dependent manner. These findings provide new insights into the B7-H3 function in the metabolic homeostasis of normal and transformed lung cancer cells, and whilst this molecule remains an interesting target for immunotherapy, these findings caution against the use of anti-B7-H3 therapies in certain clinical settings.