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Purpose:Immune checkpoint inhibitors (ICI) benefit only a minority of treated patients with cancer. Identification of biomarkers distinguishing responders and nonresponders will improve management of patients with cancer. Because the DC-HIL checkpoint differs from the PD1 pathway in expression and inhibitory mechanisms, we examined whether DC-HIL expression regulates ICI responsiveness.Experimental Design:Plasma samples were collected from patients with advanced non–small cell lung carcinoma (NSCLC) (n = 76) at baseline and/or follow-up after ICI monotherapy. Blood-soluble DC-HIL (sDC-HIL) was determined and analyzed for correlation with the early tumor response. To study the mechanisms, we measured effect of anti-DC-HIL versus anti-PDL1 mAb on growth of mouse tumor cells in experimentally metastatic lung. Influence of DC-HIL to anti-PDL1 treatment was assessed by changes in tumor response after deletion of host-DC-HIL gene, injection of DC-HIL–expressing myeloid-derived suppressor cells (MDSC), or induction of sDC-HIL expression.Results:Nonresponders expressed significantly higher levels of baseline sDC-HIL levels than responders. Among patients (n = 28) for fluctuation with time, nonresponders (14/15 cases) showed increasing or persistently elevated levels. Responders (12/13) had decreasing or persistently low levels. Among various tumors, B16 melanoma exhibited resistance to anti-PDL1 but responded to anti-DC-HIL mAb. Using B16 melanoma and LL2 lung cancer, we showed that deletion of host-derived DC-HIL expression converted the resistant tumor to one responsive to anti-PDL1 mAb. The responsive state was reversed by infusion of DC-HIL+MDSC or induction of sDC-HIL expression.Conclusions:sDC-HIL in the blood and probably DC-HIL receptor expressed by MDSC play an important role in regulating response to ICI in advanced NSCLC.

Purpose:Blocking the function of myeloid-derived suppressor cells (MDSC) is an attractive approach for cancer immunotherapy. Having shown DC-HIL/GPNMB to be the T-cell-inhibitory receptor mediating the suppressor function of MDSCs, we evaluated the potential of anti-DC-HIL mAb as an MDSC-targeting cancer treatment.Experimental Design:Patients with metastatic cancer (n = 198) were analyzed by flow cytometry for DC-HIL or PDL1 expression on blood CD14+HLA-DRno/lo MDSCs. Their suppressor function was assessed by in vitro coculture with autologous T cells, and the ability of anti-DC-HIL or anti-PDL1 mAb to reverse such function was determined. Tumor expression of these receptors was examined histologically, and the antitumor activity of the mAb was evaluated by attenuated growth of colon cancers in mice.Results:Patients with metastatic cancer had high blood levels of DC-HIL+ MDSCs compared with healthy controls. Anti-DC-HIL mAb reversed the in vitro function in ∼80% of cancer patients tested, particularly for colon cancer. Despite very low expression on blood MDSCs, anti-PDL1 mAb was as effective as anti-DC-HIL mAb in reversing MDSC function, a paradoxical phenomenon we found to be due to upregulated expression of PDL1 by T-cell-derived IFNγ in cocultures. DC-HIL is not expressed by colorectal cancer cells but by CD14+ cells infiltrating the tumor. Finally, anti-DC-HIL mAb attenuated growth of preestablished colon tumors by reducing MDSCs and increasing IFNγ-secreting T cells in the tumor microenvironment, with similar outcomes to anti-PDL1 mAb.Conclusions:Blocking DC-HIL function is a potentially useful treatment for at least colorectal cancer with high blood levels of DC-HIL+ MDSCs.See related commentary by Colombo, p. 453

Supplementary Table S1. Summary of MDSC analysis of metastatic cancer patents. aAll cancer patients are diagnosed as stage III or IV. Supplementary Table S2. Correlation of variables and expansion of DC-HIL+ MDSCs among colorectal cancer patients. Supplementary Table S3. % DC-HIL+ MDSC/PBMC in subgroups with vs. without radiotherapy. Supplementary Table S4. Reversing effects of anti-DC-HIL vs. anti-PDL1 mAb on MDSC suppressor function between two different treatments. Supplementary Table S5. DC-HIL and PDL1 expression in colorectal cancer tissues.

Supplementary Fig. S1. Early stages CRC patients expressed very low DC-HIL expression on MDSCs. Supplementary Fig. S2. No effect of anti-CD14 mAb on T cell suppressor function of MDSCs. Supplementary Fig. S3. DC-HILneg MC38 and CT26 mouse colon cancer cell lines exhibit high response to anti-DC-HIL treatment. Supplementary Fig. S4. MDSCs in spleen and draining lymph nodes did not express DC-HIL. Supplementary Fig. S5. Combination treatment of anti-DC-HIL/anti-PDL1 mAb did not produce synergistic anti-tumor effects to MC38 colon cancer. Supplementary Fig. S6. Changes in health conditions of mice treated with anti-DC-HIL mAb. Supplementary Fig. S7. Representative FACS data showing frequency of immune cells in the tumor microenvironment of mice treated with mAb. Supplementary Fig. S8. Blood granulocytic MDSC of CRC patients expressed low-levels of DC-HIL and were not immunosuppressive in ex vivo cultures.

Figure S1, Figure S2, Figure S3, Figure S4, Figure S5, Figure S6, Figure S7, Figure S8, Table S1, Table S2, Table S3

Doxorubicin (Dox) is one of the most effective drugs for the treatment of childhood cancer patients. However, there is a correlation between Dox dose/intensity and cardiotoxicity. We developed a juvenile mouse cardiotoxicity model and showed that Dox treatment affected the cardiac vessel morphology characterized by punctate vessels with decreased pericytes and endothelial cells. Cardiac blood flow and function (as quantified by echocardiography [Echo]) were also compromised. These abnormalities were not seen in mice treated with exercise (Ex) during Dox. We also demonstrated increased c-kit+ cells in the hearts of mice treated with Dox + Ex. To determine the origin of these c-kit+ cells we performed a bone marrow (BM) transplant using GFP+ BM cells from C57BL/6Tg(UBC-GFP)30Scha/J mice into C57BL/6 WT mice treated with Busulfan and anti-CD4 and anti-DC8 Abs. BM engraftment was documented 4 wks post-transplant. Transplanted mice were treated with Dox twice/wk x 2 wks; Dox + Ex, or control (no treatment). Cardiac function was assessed after treatment by Echo and the heart tissues were evaluated by immunohistochemistry (IHC) for cells expressing GFP (denoting cells derived from the BM); GFP+/NG2+ or GFP+/CD31+ (BM cells differentiated into pericytes or endothelial cells respectively). Echo data confirmed decreased cardiac function in Dox but not Dox + Ex-treated mice. Few GFP+ cells were found in the hearts from control or Dox-treated mice. By contrast, mice treated with Dox + Ex showed a significant increase in GFP+ cells indicating that exercise had induced the migration of BM cells into the heart. Co-localization of GFP/NG2 and GFP/CD31 indicated that these BM stem cells had differentiated into both endothelial cells and pericytes, and that these cells were incorporated into the cardiac vessels. Few GFP+cTnI+ cells were observed in the heart tissues from mice treated with Ex + Dox, indicating that the BM stem cells had not differentiated into cardiomyoctes. These studies suggest that acute Dox-induced cardiac damage may be partially mediated by decreased blood flow secondary to damaged vessels with decreased vascular endothelial cells and pericytes. Ex may inhibit this effect by inducing the migration of BM stem cells to repair the damaged vessels. We also showed that Dox therapy induced phosphorylation of MST1/2, LATS1 and YAP, decreased total YAP, and induced cleavage of caspase-3 and PARP. These changes were not seen in Dox + Ex hearts suggesting that Ex protected cardiomyocytes from Dox-induced apoptosis by suppressing Hippo-YAP signaling. Taken together, our data demonstrated that the protective effect of Ex may be mediated by restoring cardiac blood flow which may in turn inhibit Dox-induced Hippo-YAP signaling-mediated cardiomyocyte apoptosis. These data support the concept that exercise interventions have the potential to decrease Dox-induced cardiac damage and morbidity in childhood cancer patients. Citation Format: Rong-Hua Tao, Masato Kobayashi, Fei Wang, Yuanzheng Yang, Eugenie S. Kleinerman. Dissecting the mechanism of exercise-mediated protection of Dox-induced cardiotoxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5422.

Myeloid-derived suppressor cells (MDSC) are the most potent suppressors of T-cell function, and their exponential proliferation in cancer states counteracts the benefits of immunotherapy given to these patients. Having discovered the T cell-inhibitory DC-HIL receptor, we showed that DC-HIL is responsible for MDSC’s T-cell suppressive function. We found that melanoma patients (but not healthy controls) harbor in the blood an expanded population of DC-HIL+ MDSC, whose suppressor effects in vitro can be blocked by our 3D5 anti-DC-HIL mAb. To determine whether similar outcomes apply to non-melanoma cancers, we recruited patients with metastases from bladder (BL, n=4), breast (BR, n=11), colon (CO, n=44), kidney (KI, n=6), lung (LU, n=20), pancreatic (PA, n=17), and prostate (PR, n=9) cancer. FACS analysis of blood samples showed all cancer types (except LU) were associated with elevated blood HLA-DRno/low CD14+ MDSC vs. age-matched controls (n=21, median of 0.5%). % DC-HIL+ MDSC in PBMC was also significantly high in all cancer types; median of 2.5% for BL; 1.7% for BR; 3.5% for CO; 3.6% for KI; 0.3% for LU; 1.8% for PA; and 4.8% for PR, vs. 0.04% for healthy controls. MDSC in all cancer types showed high DC-HIL-positivity (20-90% vs. 8% for controls). We assayed T-cell suppressor activity by purifying MDSC and T-cells from the same patient and co-culturing them at varying cell ratios, with co-stimulators. Activity was determined by % suppression in IFN-γ secretion, which was correlated with % DC-HIL-positivity of MDSC. MDSC from KI (n=3) were more suppressive than those from CO and PA, with suppressor activity correlating with DC-HIL-positivity: Pearson’s r for 0.5 for CO (n=13) and 0.73 for PA (n=8). We evaluated effects of anti-DC-HIL mAb on co-cultures of MDSC/ T-cells from cancer patients (n=25). The mAb restored IFN-γ secretion by >30% in 8/15 cases of CO; 4/8 of PA, and 2/2 of KI. To determine whether DC-HIL+ MDSC migrate into cancer sites, lesional specimens from CO patients (n=5) with high blood counts of DC-HIL+ MDSC were immunohistochemically stained for CD14 and DC-HIL expression: DC-HIL was absent from all cancer cells, but present strongly in many CD14+ cells surrounding the cancers. This outcome contrasts starkly with melanoma, in which DC-HIL was expressed highly by the cancer cells surrounded by only a few DC-HIL+ CD14+ cells. Finally we evaluated in vivo effects of blocking DC-HIL using a mouse model of MC38 colon cancer, which is DC-HILneg. MC38 tumor challenge induced DC-HIL+ Gr1lowLy6Chigh MDSC in the tumor site. Intravenous infusion of anti-DC-HIL mAb into mice with pre-established MC38 tumor significantly inhibited tumor growth by 50-70%, reduced blood levels of MDSC, and enhanced cytotoxic T-lymphocyte response. Our findings support DC-HIL blockade as a potential treatment for metastatic cancers, with high blood levels of DC-HIL+ MDSC as a prognostic marker for the best responders. Citation Format: Masato Kobayashi, Jin-Sung Chung, Muhammad Beg, Yull Arriaga, Udit Verma, Kevin Courtney, John Mansour, Barbara Haley, Saad Khan, Yutaka Horiuchi, David Harker, Purva Gopal, Ponciano D. Cruz, Kiyoshi Ariizumi. Blocking the DC-HIL receptor reverses the T-cell suppression induced by proliferating myeloid-derived suppressor cells in common cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3660. doi:10.1158/1538-7445.AM2017-3660