Evaluating immunotherapeutic outcomes in triple-negative breast cancer with a cholesterol radiotracer in mice.

Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement for cholesterol in the activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in preclinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and -nonresponsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells allows detection of T cell activation and has potential to assess the success of ICI therapy.

Predicting efficacy of immunotherapy in mice with triple negative breast cancer using a cholesterol PET radiotracer.

Predicting the response to cancer immunotherapy remains an unmet challenge in triple-negative breast cancer (TNBC) and other malignancies. T cells, the major target of current checkpoint inhibitor immunotherapies, accumulate cholesterol during activation to support proliferation and signaling. The requirement of cholesterol for anti-tumor functions of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged a novel positron emission tomography (PET) radiotracer, FNP-59. FNP-59 is an analog of cholesterol that our group has validated as an imaging biomarker for cholesterol uptake in pre-clinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing immune checkpoint inhibitor (ICI)-responsive EO771 tumors to non-responsive AT-3 tumors, we found significantly higher uptake of a fluorescent cholesterol analog in T cells of the ICI-responsive tumors both in vitro and in vivo. Using the FNP-59 radiotracer, we discovered that accumulation of cholesterol by T cells increased further in ICI-responding tumors that received ant-PD-1 checkpoint immunotherapy. We verified these data by mining single cell sequencing data from patients with TNBC. Patients with tumors containing cycling T cells showed gene expression signatures of cholesterol uptake and trafficking. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells predict T cell activation and success of ICI therapy.

Effects of iron modulation on mesenchymal stem cell-induced drug resistance in estrogen receptor-positive breast cancer.

Patients with estrogen receptor-positive (ER+) breast cancer, the most common subtype, remain at risk for lethal metastatic disease years after diagnosis. Recurrence arises partly because tumor cells in bone marrow become resistant to estrogen-targeted therapy. Here, we utilized a co-culture model of bone marrow mesenchymal stem cells (MSCs) and ER+ breast cancer cells to recapitulate interactions of cancer cells in bone marrow niches. ER+ breast cancer cells in direct contact with MSCs acquire cancer stem-like (CSC) phenotypes with increased resistance to standard antiestrogenic drugs. We confirmed that co-culture with MSCs increased labile iron in breast cancer cells, a phenotype associated with CSCs and disease progression. Clinically approved iron chelators and in-house lysosomal iron-targeting compounds restored sensitivity to antiestrogenic therapy. These findings establish iron modulation as a mechanism to reverse MSC-induced drug resistance and suggest iron modulation in combination with estrogen-targeted therapy as a promising, translatable strategy to treat ER+ breast cancer.

MyD88 Costimulation in Donor CD8+ T Cells Enhances the Graft-versus-Tumor Effect in Murine Hematopoietic Cell Transplantation.

Donor-derived lymphocytes from allogeneic hematopoietic cell transplantation (allo-HCT) or donor lymphocyte infusion can mediate eradication of host tumor cells in a process labeled the graft-versus-tumor (GVT) effect. Unfortunately, these treatments have produced limited results in various types of leukemia because of an insufficient GVT effect. In this context, molecular engineering of donor lymphocytes to increase the GVT effect may benefit cancer patients. Activating MyD88 signaling in CD8+ T cells via TLR enhances T cell activation and cytotoxicity. However, systemic administration of TLR ligands to stimulate MyD88 could induce hyperinflammation or elicit protumor effects. To circumvent this problem, we devised a synthetic molecule consisting of MyD88 linked to the ectopic domain of CD8a (CD8α:MyD88). We used this construct to test the hypothesis that MyD88 costimulation in donor CD8+ T cells increases tumor control following allo-HCT in mice by increasing T cell activation, function, and direct tumor cytotoxicity. Indeed, an increase in both in vitro and in vivo tumor control was observed with CD8α:MyD88 T cells. This increase in the GVT response was associated with increased T cell expansion, increased functional capacity, and an increase in direct cytotoxic killing of the tumor cells. However, MyD88 costimulation in donor CD8+ T cells was linked to increased yet nonlethal graft-versus-host disease in mice treated with these engineered CD8+ T cells. Given these observations, synthetic CD8α:MyD88 donor T cells may represent a unique and versatile approach to enhance the GVT response that merits further refinement to improve the effectiveness of allo-HCT.

Radiotherapy Both Promotes and Inhibits Myeloid-Derived Suppressor Cell Function: Novel Strategies for Preventing the Tumor-Protective Effects of Radiotherapy.

Cancer immunotherapies aimed at neutralizing the programmed death-1 (PD-1) immune suppressive pathway have yielded significant therapeutic efficacy in a subset of cancer patients. However, only a subset of patients responds to antibody therapy with either anti-PD-1 or anti-PD-L1 antibodies. These patients appear to have so-called "hot" tumors containing tumor-reactive T cells. Therefore, checkpoint blockade therapy may be effective in a larger percentage of cancer patients if combined with therapeutics that also activate tumor-reactive T cells. Radiotherapy (RT) is a prime candidate for combination therapy because it facilitates activation of both local antitumor immunity and antitumor immunity at non-radiated, distant sites (abscopal response). However, RT also promotes tumor cell expression of PD-L1 and facilitates the development of myeloid-derived suppressor cells (MDSC), a population of immune suppressive cells that also suppress through PD-L1. This article will review how RT induces MDSC, and then describe two novel therapeutics that are designed to simultaneously activate tumor-reactive T cells and neutralize PD-1-mediated immune suppression. One therapeutic, a CD3xPD-L1 bispecific T cell engager (BiTE), activates and targets cytotoxic T and NKT cells to kill PD-L1+ tumor cells, despite the presence of MDSC. The BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma tumors. The second therapeutic is a soluble form of the costimulatory molecule CD80 (sCD80). In addition to costimulating through CD28, sCD80 inhibits PD-1 suppression by binding to PD-L1 and sterically blocking PD-L1/PD-1 signaling. sCD80 increases tumor-infiltrating T cells and significantly extends survival time of mice carrying established, syngeneic tumors. sCD80 does not suppress T cell function via CTLA-4. These studies suggest that the CD3xPD-L1 BiTE and sCD80 may be efficacious therapeutics either as monotherapies or in combination with other therapies such as radiation therapy for the treatment of cancer.

CD3xPDL1 bi-specific T cell engager (BiTE) simultaneously activates T cells and NKT cells, kills PDL1+ tumor cells, and extends the survival of tumor-bearing humanized mice.

Bi-specific T cell engagers (BiTEs) activate T cells through CD3 and target activated T cells to tumor-expressed antigens. BiTEs have shown therapeutic efficacy in patients with liquid tumors; however, they do not benefit all patients. Anti-tumor immunity is limited by Programmed Death 1 (PD1) pathway-mediated immune suppression, and patients who do not benefit from existing BiTES may be non-responders because their T cells are anergized via the PD1 pathway. We have designed a BiTE that activates and targets both T cells and NKT cells to PDL1+ cells. In vitro studies demonstrate that the CD3xPDL1 BiTE simultaneously binds to both CD3 and PDL1, and activates healthy donor CD4+ and CD8+ T cells and NKT cells that are specifically cytotoxic for PDL1+ tumor cells. Cancer patients' PBMC are also activated and cytotoxic, despite the presence of myeloid-derived suppressor cells. The CD3xPDL1 BiTE significantly extends the survival time and maintains activated immune cell levels in humanized NSG mice reconstituted with human PBMC and carrying established human melanoma tumors. These studies suggest that the CD3xPDL1 BiTE may be efficacious for patients with PDL1+ solid tumors, in combination with other immunotherapies that do not specifically neutralize PD1 pathway-mediated immune suppression.