Erythropoietin (EPO), used to treat anemia in cancer patients, has been reported to accelerate tumor progression and increase mortality. Research of the mechanism for this effect has focused upon EPOR expression by tumor cells. We model the high macrophage to lymphocyte ratio found in tumor microenvironments (TMEs) by culturing peritoneal cavity (PerC) cells that naturally have a high macrophage to T cell ratio. Following TCR ligation, C57BL/6J PerC T cell proliferation is suppressed due to IFNγ-triggered inducible nitric oxide synthase (iNOS) expression. EPO was tested in the PerC culture model and found to increase T cell suppression. This effect could be abrogated by inhibiting iNOS by enzyme inhibition, genetic ablation, or blocking IFNγ signaling. Flow cytometry revealed the EPOR on CD11b+F4/80+ macrophages. These results suggest that EPO could increase T cell suppression in the TME by acting directly on macrophages.

Tumors are often comprised of microenvironments (TMEs) with a high proportion of cells and molecules that regulate immunity. Peritoneal cavity (PerC) cell culture reproduces key features of TMEs as lymphocyte proliferation is suppressed by PerC macrophages (Mϕs). We monitored the expression of T cell stimulatory (Class II MHC, B7) and inhibitory (PD-L1) molecules by PerC APCs before and after culture and report here that IFNγ-driven PD-L1 expression increased markedly on PerC Mϕs after TCR ligation, even more so than seen with direct APC activation by LPS. Considering the high APC composition of and pronounced PD-L1 expression by PerC cells, it was surprising that blocking PD-1/PD-L1 interaction by mAb neutralization or genetic ablation did not relieve suppression. This result parallels TME challenges observed in the clinic and validates the need for further study of this culture model to inform strategies to promote anti-tumor immunity.

Early biomarkers of ovarian cancer (OvCa) are needed for timely disease detection. Monitoring alterations in the immune system might be informative in this regard. To study humoral immunity during OvCa, we transplant murine epithelial carcinoma cells (ID8) into the peritoneal cavity (PerC). As the PerC is enriched for B1 cells, we assessed B cell subset composition via flow cytometry. As OvCa developed PerC B1 cells were lost while B2 cells persisted in the PerC and spleen (SP). This difference might reflect that self-reactive B1 cells have a role in housekeeping and lack progenitors in adult mice. We tested the latter hypothesis by monitoring transitional (T) B cells, and found the T1, T2, and T3 subsets were markedly reduced in late stage disease, suggesting depletion of B1 B cells, as opposed to expansion of B2 B cells. Intriguingly, marginal zone B cells (MZB) expanded early then contracted as OvCa progressed. We speculate that housekeeping burden grows with OvCa progression and the cells most associated with this vital homeostatic process, notably B1 and MZB cells, are most at risk. These findings highlight the disruption of B cell biology in mice challenged with OvCa, and may help to inform strategies to develop biomarkers for the disease.

The high myeloid to lymphoid ratio of cultured peritoneal cavity (PerC) leukocytes can serve as an in vitro model to study the tumor microenvironment (TME). C57BL/6J PerC T cell responses to TCR ligation (anti-CD3) and mitogen (ConA) are suppressed in these cultures. Likewise, the PerC B cell response to BCR (F[ab’2] anti-IgM) and TLR4 (TLR4L/LPS) ligation are suppressed. T cell suppression can be liberated by neutralizing IFNγ or by blocking iNOS with 1-methyl arginine. The BCR response is recovered by blocking prostaglandin production with indomethacin; the LPS response by neutralizing IL10. To dissect putative receptor-ligand signals in this model, we investigated expression of the inhibitory cell surface marker PDL1/B7H1/CD274 on PerC cells. TCR ligation increased PDL1 expression on macrophages (Mfs), B1, and B2 cells and was reduced by blocking IFNγ. TLR4 ligation increased PDL1 expression on Mfs and both B cell subsets and was reduced by blocking IFNAR1. Interestingly, BCR ligation increased B7H1 and Class II expression on Mfs and B2 cells, but not B-1 cells. Increased B7H1 expression could be reduced by neutralizing IL6, IFNβ, IFNγ, or IFNAR1. TCR ligation led to B1, but not B2, cell division and proliferating B cells increased IL10 production. These findings illustrate that TCR, and surprisingly BCR, ligation can foster immune suppression in macrophage-dense cultures and suggest that regulatory B cells must be considered when designing immunomodulatory cancer therapies.

We have been studying the emergence of myeloid-derived suppressor cells (MDSCs) following i.p. transfer of ID8 cells (mouse ovarian surface epithelial cell carcinoma) into intact, syngeneic, C57BL/6J (wild type, WT) mice. Our current work studies the impact of the cytokine (CK) environment on ID8 expansion and MDSC emergence in the peritoneal cavity (PerC). We wanted to determine if particular pro- (IFNg) or anti- (IL4, IL10) inflammatory CKs were drivers of disease in this model. We found that, relative to WT mice, the development of hemorrhagic ascites was accelerated in IL10-/-, IL4-/-, and IFNgR-/- mice. The greatest percentage and number of MDSCs was observed in IL10-/- mice, which also had the greatest expansion of ID8 cells. ID8 triggered a marked increase in PerC Mfs, particularly in the IL10-/- mice, and lymphocytes, particularly in the IFNgR-/- and IL4-/- mice. However, for all mice, the percent representation of B cells in the total PerC cell pool declined markedly, particularly in the IL10-/- mice (> 70%). Interestingly, beyond the peritoneal locus of disease, both B and T lymphocyte percentages dropped in the spleens of all mice. This was seen without a concomitant increase in the number of MDSCs or appearance of ID8 in the spleen. These observations reinforce that inflammation drives MDSC emergence and suggest that changes in lymphocyte populations could serve as biomarkers for assessing ovarian cancer.