Macrophages as a Therapeutic Target in Metastatic Prostate Cancer: A Way to Overcome Immunotherapy Resistance?

Prostate cancer (PC) is the most common malignancy and the fifth cause of cancer death in men. The treatment for localized or locally advanced stages offers a high probability of cure. Even though the therapeutic landscape has significantly improved over the last decade, metastatic PC (mPC) still has a poor prognosis mainly due to the development of therapy resistance. In this context, the use of immunotherapy alone or in combination with other drugs has been explored in recent years. However, T-cell directed immune checkpoint inhibitors (ICIs) have shown limited activity with inconclusive results in mPC patients, most likely due to the highly immunosuppressive PC tumor microenvironment (TME). In this scenario, targeting macrophages, a highly abundant immunosuppressive cell type in the TME, could offer a new therapeutic strategy to improve immunotherapy efficacy. In this review, we summarize the growing field of macrophage-directed immunotherapies and discuss how these could be applied in the treatment of mPC, focusing on their combination with ICIs.

EXTH-13. LOCAL DELIVERY OF AN IL-15 SUPERAGONIST USING A REPLICATING RETROVIRUS SIGNIFICANTLY IMPROVES SURVIVAL AND LYMPHOCYTE INFILTRATION IN POORLY IMMUNOGENIC MURINE GLIOBLASTOMA MODELS

Glioblastoma (GBM) leads to severe systemic and local immunosuppression, and immunotherapies have had limited clinical success. Here, we evaluated the treatment efficacy of RLI, a superagonist of T-cell activator IL-15, delivered to tumor cells using a tumor-selective retroviral replicating vector (RRV) in the syngeneic murine SB28 and Tu2449 GBM models, which are both engineered to be poorly immunogenic with low-mutational burden and known resistance to immunotherapy, and hence more accurate biomimetic models of human GBM. RRV-RLI replicated and spread effectively in cultured murine GBM cells with robust production of functional RLI (165.4 ± 5.3 ng/mL). Stereotactic injection of RRV-RLI into pre-established intracerebral SB28 tumors significantly reduced tumor growth on bioluminescent imaging, and increased median survival compared to control mice (55 vs. 19 days, p=0.002), leading to long-term survival in 12% of treated mice. In the Tu2449 model, imaging results showed complete eradication of intracerebral tumors after RRV-RLI treatment, with long-term survival (median not reached) in > 85% of treated mice, compared to a median survival of 12.5 days in control mice (p=0.001). RRV-RLI treated tumors showed significantly increased CD8 T-cell infiltration, without altering immunosuppressive cell populations. Similarly, broad anti-tumor inflammatory changes, including increased expression of genes involved in T-cell activation and killing, were observed in the NanoString nCounter platform using a 770-gene panel representing various immune cell types. Notably, RLI was not detected in the blood of treated mice, and tumor-localized RRV-RLI gene delivery showed no adverse systemic immune effects in either model. In summary, RRV-mediated RLI immunotherapy results in immunostimulatory and pro-inflammatory changes to the tumor microenvironment and achieves a significant survival benefit in two poorly immunogenic syngeneic murine models of GBM. This tumor-localized immunomodulatory gene therapy has the potential to safely reverse the T-cell depleted immunophenotype of GBM.

616 CD47 blockade modulates immunosuppressive checkpoint molecules and cellular metabolism to sensitize triple-negative breast cancer tumors to immune checkpoint blockade therapy

BackgroundTriple-negative breast cancer(TNBC) lacks druggable targets and has high metastatic incidence. Immune checkpoint blockades (ICB) are FDA approved for TNBC treatment, but therapeutic response and biomarkers are limited. CD47 is an integral membrane protein overexpressed on cancer cells that alters anti-tumor immunosurveillance, resulting in tumor progression. CD47 is involved in metabolic reprogramming but whether CD47 is a marker of progression and its role in ICB response for TNBC remains unknown.MethodsHuman TNBC biopsies were subjected to immunohistochemical analysis to determine CD47 role in TNBC progression. To determine CD47 impact on tumor burden, a carcinogen-induced TNBC model was performed in female wild type(WT) and cd47 null(cd47-/-) C57Bl/6 mice. To evaluate immune infiltrate signaling, tumors underwent spatial tissue proteomics by multiplexing photo-cleavable antibodies in Formalin-Fixed Paraffin-Embedded samples. An orthotopic EMT-6 murine TNBC model was performed to investigate tumor burden for CD47 monotherapy or in combination with anti-PD-L1 therapy.ResultsHuman matched primary, and metastatic TNBC biopsies increased immunoreactivity to CD47, signifying a potential therapeutic target(n=24). CD47 deficiency in the carcinogen-induced DMBA model decreased tumor incidence, weight, and area compared to WT(n=8/group,*p<0.003). Since CD47 can regulate metabolism, tumors underwent metabolomic analysis. Principal component analysis displayed differentially regulated metabolites between WT and cd47-/- tumors. Decreased carnitine conjugated fatty acids and ketone bodies were observed in cd47-/- tumors compared to WT, suggesting decreased fatty acid availability and/or metabolism(n=9/group,*p<0.05). TNBC cell respiratory measurements validated that targeting CD47 shifted metabolic dependency from fatty acid oxidation to glycolysis(n=3,*p<0.05). Kynurenine/tryptophan pathway metabolites, which catabolize Indoleamine-2,3-dioxygenase(IDO1) and involved in anti-PD-1/PD-L1 resistance, were decreased in cd47-/- tumors compared to WT(n=9/group,*p<0.05). Spatial proteomic analysis determined that cd47-/- tumors had elevated immune cell infiltration(CD45+, CD3+), suggesting CD47 absence enhances tumor immunogenicity and immune-mediated tumor ablation. Multiplexing of photo-cleavable antibodies increased protein expression of immune checkpoint molecules(PD-L1,VISTA,B7-H3,BatF3) and immunosuppressive cell types(CD11b+,Ly6c+) in WT tumors compared to cd47-/-, suggesting CD47 absence limits immunosuppressive signaling(n=16/group,*p<0.05). Since anti-PD-L1 therapies are approved to treat TNBC and WT tumors have PD-L1 upregulation, we examined how targeting CD47 would impact tumor burden of mice receiving anti-PD-L1 therapy. Targeting CD47 or PD-L1 as monotherapy decreased tumor burden; however, in combination it further reduced tumor burden compared to anti-PD-L1 treatment due to increased intratumoral granzyme B secreting cytotoxic T cells(n=4–8/group,*p<0.05).ConclusionsOur data indicates that CD47 may serve as a marker of anti-PD-L1 response, and targeting CD47 enhances immunogenicity and decreases immunosuppressive molecules, sensitizing TNBC tumors to anti-PD-L1 therapy to reduce tumor burden.AcknowledgementsDSP is supported by the NCI R21 (CA249349) and the American Cancer Society Research Scholar Grant (133727-RSG-19-150-01-LIB). ERS is supported by the NIAID Immunology and Pathogenesis T32 Training Grant (T32AI007401).Ethics ApprovalAnimal studies were approved by the Institutional Care and Use Committee, Wake Forest Health Sciences.

Transcriptomic-Based Identification of the Immuno-Oncogenic Signature of Cholangiocarcinoma for HLC-018 Multi-Target Therapy Exploration

Cholangiocarcinomas (CHOLs), hepatobiliary malignancies, are characterized by high genetic heterogeneity, a rich tumor microenvironment, therapeutic resistance, difficulty diagnosing, and poor prognoses. Current knowledge of genetic alterations and known molecular markers for CHOL is insufficient, necessitating the need for further evaluation of the genome and RNA expression data in order to identify potential therapeutic targets, clarify the roles of these targets in the tumor microenvironment, and explore novel therapeutic drugs against the identified targets. Consequently, in our attempt to explore novel genetic markers associated with the carcinogenesis of CHOL, five genes (SNX15, ATP2A1, PDCD10, BET1, and HMGA2), collectively termed CHOL-hub genes, were identified via integration of differentially expressed genes (DEGs) from relatively large numbers of samples from CHOL GEO datasets. We further explored the biological functions of the CHOL-hub genes and found significant enrichment in several biological process and pathways associated with stem cell angiogenesis, cell proliferation, and cancer development, while the interaction network revealed high genetic interactions with a number of onco-functional genes. In addition, we established associations between the CHOL-hub genes and tumor progression, metastasis, tumor immune and immunosuppressive cell infiltration, dysfunctional T-cell phenotypes, poor prognoses, and therapeutic resistance in CHOL. Thus, we proposed that targeting CHOL-hub genes could be an ideal therapeutic approach for treating CHOLs, and we explored the potential of HLC-018, a novel benzamide-linked small molecule, using molecular docking of ligand-receptor interactions. To our delight, HLC-018 was well accommodated with high binding affinities to binding pockets of CHOL-hub genes; more importantly, we found specific interactions of HLC-018 with the conserved sequence of the AT-hook DNA-binding motif of HMGA2. Altogether, our study provides insights into the immune-oncogenic phenotypes of CHOL and provides valuable information for our ongoing experimental validation.

CD177 modulates the function and homeostasis of tumor-infiltrating regulatory T cells

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.

The Immunomodulatory Effect of Triptolide on Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are known to have immunosuppressive ability and have been used in clinical treatment of acute graft-versus-host disease, one of severe complications of the hematopoietic stem cell transplantation. However, MSCs are activated to suppress the immune system only after encountering an inflammatory stimulation. Thus, it will be ideal if MSCs are primed to be activated and ready to suppress the immune reaction before being administered. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb-Tripterygium wilfordii Hook.f. It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aimed to use TPL to prime umbilical cord-derived MSCs (TPL-primed UC-MSCs) to enter a stronger immunosuppressive status. UC-MSCs were primed with TPL, which was washed out thoroughly, and the TPL-primed UC-MSCs were resuspended in fresh medium. Although TPL inhibited the proliferation of UC-MSCs, 0.01 μM TPL for 24 h was tolerable. The surface markers of TPL-primed UC-MSCs were identical to those of non-primed UC-MSCs. TPL-primed UC-MSCs exhibited stronger anti-proliferative effect for activated CD4+ and CD8+ T cells in the allogeneic mixed lymphocyte reaction assay than the non-primed UC-MSCs. TPL-primed UC-MSCs promoted the expression of IDO-1 in the presence of IFN-γ, but TPL alone was not sufficient. Furthermore, TPL-primed UC-MSCs showed increased expression of PD-L1. Conclusively, upregulation of IDO-1 in the presence of IFN-γ and induction of PD-L1 enhances the immunosuppressive potency of TPL-primed UC-MSCs on the proliferation of activated T cells. Thus, TPL- primed MSCs may provide a novel immunosuppressive cell therapy.

CCL2: An Important Mediator Between Tumor Cells and Host Cells in Tumor Microenvironment

Tumor microenvironment (TME) formation is a major cause of immunosuppression. The TME consists of a considerable number of macrophages and stromal cells that have been identified in multiple tumor types. CCL2 is the strongest chemoattractant involved in macrophage recruitment and a powerful initiator of inflammation. Evidence indicates that CCL2 can attract other host cells in the TME and direct their differentiation in cooperation with other cytokines. Overall, CCL2 has an unfavorable effect on prognosis in tumor patients because of the accumulation of immunosuppressive cell subtypes. However, there is also evidence demonstrating that CCL2 enhances the anti-tumor capability of specific cell types such as inflammatory monocytes and neutrophils. The inflammation state of the tumor seems to have a bi-lateral role in tumor progression. Here, we review works focusing on the interactions between cancer cells and host cells, and on the biological role of CCL2 in these processes.

The pre-metastatic niche in lymph nodes: formation and characteristics

Lymph node metastasis is a crucial prognostic parameter in many different types of cancers and a gateway for further dissemination to distant organs. Prior to metastatic dissemination, the primary tumor prepares for the remodeling of the draining (sentinel) lymph node by secreting soluble factors or releasing extracellular vesicles that are transported by lymphatic vessels. These important changes occur before the appearance of the first metastatic cell and create what is known as a pre-metastatic niche giving rise to the subsequent survival and growth of metastatic cells. In this review, the lymph node structure, matrix composition and the emerging heterogeneity of cells forming it are described. Current knowledge of the major cellular and molecular processes associated with nodal pre-metastatic niche formation, including lymphangiogenesis, extracellular matrix remodeling, and immunosuppressive cell enlisting in lymph nodes are additionally summarized. Finally, future directions that research could possibly take and the clinical impact are discussed.

Multiplexed 3D atlas of state transitions and immune interactions in colorectal cancer

Advanced solid cancers are complex assemblies of tumor, immune, and stromal cells that invade adjacent tissue and spread to distant sites. Here we use highly multiplexed tissue imaging, spatial statistics, and machine learning to identify cell types and states underlying morphological features of known diagnostic and prognostic significance in colorectal cancer. We find that a thorough spatial analysis requires imaging the entire tumor region, not small fields of view (e.g. those found in tissue microarrays). When this condition is met, the data reveal frequent transitions between histological archetypes (tumor grades and morphologies) correlated with molecular gradients. At the tumor invasive margin, where tumor, normal, and immune cells compete, localized features in 2D such as tumor buds and mucin pools are seen in 3D to be large connected structures having continuously varying molecular properties. Immunosuppressive cell-cell interactions also exhibit graded variation in type and frequency. Thus, whereas scRNA-Seq emphasizes discrete changes in tumor state, whole-specimen imaging reveals the presence of large- and small-scale spatial gradients analogous to those in developing tissues.