Transmissible endoplasmic reticulum (ER) stress is emerging as a cell non-autonomous mechanism that drives the failure of immune competency within the tumor microenvironment (TME). Reprogramming of myeloid cell infiltrate within the TME to a mixed pro-inflammatory/immune suppressive phenotype is at the root of this immune dysregulation, and we show here that the unfolded protein response (UPR) is a possible origin of these events. The inositol-requiring enzyme 1 (IRE1a) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the upregulation of IL-6, IL-23, Arginase1, as well as surface expression of CD86 and PD-L1. Macrophages in which the IRE1a-Xbp1 axis is blocked pharmacologically or deleted genetically have significantly reduced polarization, CD86 and PD-L1 expression. Mice with IRE1a- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. RNASeq analysis showed that bone marrow derived macrophages with IRE1a deletion lose the integrity of the gene connectivity characteristic of regulated IRE1a-dependent decay (RIDD) and fail to activate PD-L1 gene expression. Thus, the IRE1a-Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immunosurveillance.

The tumor microenvironment is harbor to a variety of insults that privilege tumor cells to co-opt host immunity. Many of these insults, including nutrient deprivation and hypoxia, create endoplasmic reticulum (ER) stress for resident tumor cells. Emerging reports also suggest that ER stress in infiltrating myeloid cells leads to the immune dysfunction, rendering these inept in the eliminating the tumor and instead facilitative of tumor growth. We previously reported that this polarization may be mediated by factors derived from tumor cells undergoing ER stress that target immune infiltrating cells to then similarly undergo ER stress, termed transmissible ER stress (TERS).

This dissertation advances the findings of TERS through three central aims. In Chapter 2, I probe the role of TERS on cancer cells using a prostate cancer cell model. My findings reveal that TERS experienced cancer cells gain cytoprotection over tumor microenvironmental challenges as well as chemotherapies, which was centrally mediated by the PERK arm of the UPR. Chapter 3 reports that myeloid cells infiltrating the tumor microenvironment undergo a polarization consistent with those affected by TERS. Chemical inhibition of IRE1α during TERS led to dramatic reduction in TERS driven inflammation, immune-suppression, and angiogenesis. Finally, Chapter 4 elucidates the identity of the molecules responsible for TERS. I found TERS activity is assignable to lipid soluble molecules. Lipidomic analysis identified TERS contained enriched eicosanoids, which could not cause ER stress but drive immune suppressive features. The ER stress inducing factor behind TERS is a novel mono-unsaturated fatty acid that caused increases in ER stress, pro-inflammation, and angiogenesis in myeloid cells as well as drove the surface expression of CD86 and PD-L1.

These lines provide the elucidation of the novel factors tumor cells undergoing ER stress secrete to promote immune evasion. These results identify potential novel biomarkers of the tumor microenvironment as well as emphasize that targeting the UPR, and specifically the IRE1α axis, may serve an important target to bolster anti-tumor immunity.

Aneuploidy, aberrant chromosome numbers, is a chromosome alteration that is associated with poor prognosis and outcome in cancer. However, the exact role aneuploidy plays in cancer progression and local immune cell regulation are a matter of debate. Here we investigate the hypothesis that aneuploidy in cancer cells triggers an ER stress that will be transmitted to T cell, ultimately driving their dysfunction. We found that the conditioned medium (CM) of aneuploid murine B16.F10 and human SKOV3 cancer cells caused diminished Interferon-gamma (IFNG) and Granzyme B (GzmB) expression in T cells activated by anti-CD3/CD28 stimulation. However, the induction of aneuploidy in cancer cells did not result in UPR elevated enough to enable the transmission of UPR phenotype in T cells. T cells with diminished cytokine expression in response to aneuploid CM did not have an increase in UPR gene expression and the addition of UPR inhibitors could not restore IFNG or Granzyme expression. In fact, the induction of UPR, both through chemical means and transmissible ER stress factors, enhanced the function and activation of T cells rather than impairing their function. Finally, accumulating evidence indicates that glucose-restriction can contribute to the loss of T cell effector function by repressing EZH2, a subunit of Polycomb Repressive Complex 2 (PRC2). T cells made dysfunctional by aneuploid CM had significantly lower EZH2 expression which positively correlated with IFNG. However, addition of glucose to reactivate EZH2-mediate glucose metabolism only partially restored IFNG production in T cells. A provisional conclusion is that aneuploidy in cancer cells can cause T cell dysfunction in a cell-nonautonomous manner, but the mechanism behind it does not seem to depend on ER stress and glucose deprivation only provides a partial explanation. Further investigation is still needed to fully understand the molecular mechanism by which aneuploid CM impairs T cell immunity.

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