Metastatic cancer disease is the major cause of death in cancer patients. Because those small secondary tumors are clinically hardly detectable in their early stages, little is known about drug biodistribution and permeation into those metastatic tumors potentially contributing to insufficient clinical success against metastatic disease. Our recent studies indicated that breast cancer liver metastases may have compromised perfusion of intratumoral capillaries hindering the delivery of therapeutics for yet unknown reasons. To understand the microcirculation of small liver metastases, we have utilized computational simulations to study perfusion and oxygen concentration fields in and around the metastases smaller than 700 µm in size at the locations of portal vessels, central vein, and liver lobule acinus. Despite tumor vascularization, the results show that blood flow in those tumors can be substantially reduced indicating the presence of inadequate blood pressure gradients across tumors. A low blood pressure may contribute to the collapsed intratumoral capillary lumen limiting tumor perfusion that phenomenologically corroborates with our previously published in vivo studies. Tumors that are smaller than the liver lobule size and originating at different lobule locations may possess a different microcirculation environment and tumor perfusion. The acinus and portal vessel locations in the lobule were found to be the most beneficial to tumor growth based on tumor access to blood flow and intratumoral oxygen. These findings suggest that microcirculation states of small metastatic tumors can potentially contribute to physiological barriers preventing efficient delivery of therapeutic substances into small tumors.

We sought to determine if Stephen Paget’s “seed and soil” hypothesis of organ-preference patterns of cancer metastasis can explain the development of heterogeneity in a tumor microenvironment (TME) as well as immunotherapeutic delivery and efficacy. We established single-cell-derived clones (clones 1 and 16) from parental 4T1 murine breast cancer cells to create orthotopic primary and liver metastasis models to deconvolute polyclonal complexity cancer cells and the difference in TME-derived heterogeneities. Tumor-bearing mice were treated with anti-PD-L1 IgG or a control antibody, and immunofluorescent imaging and quantification were then performed to evaluate the therapeutic efficacy on tumor growth, the delivery of therapy to tumors, the development of blood vessels, the expression of PD-L1, the accumulation of immune cells, and the amount of coagulation inside tumors. The quantification showed an inverse correlation between the amount of delivered therapy and therapeutic efficacy in parental-cell-derived tumors. In contrast, tumors originating from clone 16 cells accumulated a significantly greater amount of therapy and responded better than clone-1-derived tumors. This difference was greater when tumors grew in the liver than the primary site. A similar trend was found in PD-L1 expression and immune cell accumulation. However, the change in the number of blood vessels was not significant. In addition, the amount of coagulation was more abundant in clone-1-derived tumors when compared to others. Thus, our findings reconfirmed the seed- and soil-dependent differences in PD-L1 expression, therapeutic delivery, immune cell accumulation, and tumor coagulation, which can constitute a heterogeneous delivery and response of immunotherapy in polyclonal tumors growing in different organs.