Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-
tumor responses. Appropriate localization and function of these cells within
tumors is critical for protective immunity, with CD8 T cell infiltration being a
biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among
cancer types, individuals, and within individual
tumors. While it is recognized that TMEs play a pivotal role in
disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with
immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical
cancer models, the MC38 colorectal and KPC pancreatic murine
tumors engineered to express human
carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed
tumors or after
immunotherapy with a CEA T cell bispecific (
CEA-TCB) surrogate antibody and anti-PD-L1 treatment.
CEA-TCB mono and combination
immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1-PD1+ effector T cells and with more minor increases in TCF1+PD1+ resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII+ macrophages, but largely avoided the deeper
tumor nest regions composed of
cancer cells and non-activated macrophages. These myeloid cell - T cell aggregates were found in close proximity to
tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after
CEA-TCB therapy, with its relative abundance positively associated with response to
therapy. Together, these studies demonstrate the utility of advanced spatial analysis in
cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within
tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.