Antibody-based immune checkpoint blockade therapy has revolutionized the field of cancer immunotherapy, yet its efficacy remains limited in immunologically cold tumors. Combining checkpoint inhibitors with costimulatory agonists improves tumoricidal activity of T cells but also can lead to off-target hepatotoxicity. Although bispecific antibodies confer tumor selectivity to alleviate undesirable adverse effects, toxicity concerns persist with increased dosing. In this issue of Cancer Research, Yuwen and colleagues introduce ATG-101, a tetravalent PD-L1×4-1BB bispecific antibody with high programmed death ligand 1 (PD-L1) affinity and low 4-1BB affinity, aiming to mitigate hepatotoxicity. ATG-101 demonstrates PD-L1-dependent 4-1BB activation, leading to selective T-cell activation within the tumor microenvironment. ATG-101 exhibits potent antitumor activity, even in large, immunologically cold, and monotherapy-resistant tumor models. Single-cell RNA sequencing reveals significant shifts of immune cell populations in the tumor microenvironment from protumor to antitumor phenotypes following ATG-101 treatment. In cynomolgus monkeys, no serious cytokine storm and hepatotoxicity are observed after ATG-101 treatment, indicating a broad therapeutic window for ATG-101 in cancer treatment. This study highlights the potential of tetravalent bispecific antibodies in cancer immunotherapy, with implications for various antibody-based treatment modalities across different fields. See related article by Yuwen et al., p. 1680.

Nutrient stress accompanies several stages of tumor progression, including metastasis formation. Metabolic reprogramming is a hallmark of cancer, and it has been associated with stress tolerance and anchorage-independent cell survival. Adaptive responses are required to support cancer cell survival under these conditions. In this issue of Cancer Research, Nam and colleagues showed that the extracellular matrix (ECM) receptor integrin β3 was upregulated in lung cancer cells in response to nutrient starvation, resulting in increased cell survival that was independent from ECM binding. Delving into the molecular mechanisms responsible for this, the authors found that integrin β3 promoted glutamine metabolism and oxidative phosphorylation (OXPHOS) by activating a Src/AMPK/PGC1α signaling pathway. Importantly, in vivo experiments confirmed that OXPHOS inhibition suppressed tumor initiation in an orthotopic model of lung cancer, while β3 knockout completely abrogated tumor initiation. These observations indicate that targeting signaling pathways downstream of αvβ3 could represent a promising therapeutic avenue to prevent lung cancer progression and metastasis. See related article by Nam et al., p. 1630.

Peripheral T cell lymphoma (PTCL) is a heterogeneous and aggressive disease with a poor prognosis. Histone deacetylase (HDAC) inhibitors have shown inhibitory effects on PTCL. A better understanding of the therapeutic mechanism underlying the effects of HDAC inhibitors could help improve treatment strategies. Here, we found that high expression of HDAC3 is associated with poor prognosis in PTCL. HDAC3 inhibition suppressed lymphoma growth in immunocompetent mice but not in immunodeficient mice. HDAC3 deletion delayed the progression of lymphoma, reduced the lymphoma burden in the thymus, spleen, and lymph nodes, and prolonged the survival of mice bearing MNU-induced lymphoma. Furthermore, inhibiting HDAC3 promoted the infiltration and enhanced the function of natural killer (NK) cells. Mechanistically, HDAC3 mediated ATF3 deacetylation, enhancing its transcriptional inhibitory activity. Targeting HDAC3 enhanced CXCL12 secretion through an ATF3-dependent pathway to stimulate NK cell recruitment and activation. Finally, HDAC3 suppression improved the response of PTCL to conventional chemotherapy. Collectively, this study provides insights into the mechanism by which HDAC3 regulates ATF3 activity and CXCL12 secretion, leading to immune infiltration and lymphoma suppression. Combining HDAC3 inhibitors with chemotherapy may be a promising strategy for treating PTCL. Key words: Histone deacetylases (HDACs), Natural killer (NK) cells, Peripheral T cell lymphoma (PTCL).

Metabolic reprogramming is a hallmark of cancer. In addition to metabolic alterations in the tumor cells, multiple other metabolically active cell types in the tumor microenvironment (TME) contribute to the emergence of a tumor-specific metabolic milieu. Here, we defined the metabolic landscape of the TME during progression of head and neck squamous cell carcinoma (HNSCC) by performing single-cell RNA sequencing (scRNA-seq) on 26 human patient specimens, including normal tissue, pre-cancerous lesions, early-stage cancer, advanced-stage cancer, lymph node metastases, and recurrent tumors. The analysis revealed substantial heterogeneity at the transcriptional, developmental, metabolic, and functional levels in different cell types. SPP1+ macrophages were identified as a pro-tumor and pro-metastatic macrophage subtype with high fructose and mannose metabolism, which was further substantiated by integrative analysis and validation experiments. An inhibitor of fructose metabolism reduced the proportion of SPP1+ macrophages, reshaped the immunosuppressive TME, and suppressed tumor growth. In conclusion, this work delineated the metabolic landscape of HNSCC at a single-cell resolution and identified fructose metabolism as a key metabolic feature of a pro-tumor macrophage subpopulation.

Circadian clock perturbation frequently occurs in cancer and facilitates tumor progression by regulating malignant growth and shaping the immune microenvironment. Emerging evidence has indicated that clock genes are disrupted in melanoma and linked to immune escape. Here, we found that the expression of retinoic acid receptor-related orphan receptor-α (RORA) is downregulated in melanoma patients and that patients with higher RORA expression have a better prognosis after immunotherapy. Additionally, RORA was significantly positively correlated with T-cell infiltration and recruitment. Overexpression or activation of RORA stimulated cytotoxic T-cell-mediated antitumor responses. RORA bound to the CD274 promoter and formed an inhibitory complex with HDAC3 to suppress PD-L1 expression. In contrast, the DEAD-box helicase family member DDX3X competed with HDAC3 for binding to RORA, and DDX3X overexpression promoted RORA release from the suppressive complex and thereby increased PD-L1 expression to generate an inhibitory immune environment. The combination of a RORA agonist with an anti-CTLA4 antibody synergistically increased T-cell antitumor immunity in vivo. A score based on the combined expression of HDAC3, DDX3X and RORA correlated with immunotherapy response in melanoma patients. Together, this study elucidates a mechanism of clock component-regulated antitumor immunity, which will help inform the use of immunotherapy and lead to improved outcomes for melanoma patients receiving combined therapeutic treatments.

Pulmonary delivery of immunostimulatory agents such as poly(I:C) to activate double-stranded RNA sensors MDA5 and RIG-I within lung-resident antigen-presenting cells is a potential strategy to enhance antitumor immunity by promoting type I interferon secretion. However, following pulmonary delivery, poly(I:C) suffers from rapid degradation and poor endosomal escape, thus limiting its potency. Inspired by the structure of a virus that utilizes internal viral proteins to tune the loading and cytosolic delivery of viral nucleic acids, we developed a liponanogel (LNG)-based platform to overcome the delivery challenges of poly(I:C). The LNG consisted of an anionic polymer hyaluronic acid-based nanogel core coated by a lipid shell, which served as a protective layer to stabilize the nanogel core in the lungs. The nanogel core was protonated within acidic endosomes to enhance the endosomal membrane permeability and cytosolic delivery of poly(I:C). After pulmonary delivery, LNG-poly(I:C) induced 13.7-fold more IFNβ than poly(I:C) alone and 2-fold more than poly(I:C) loaded in the state-of-art lipid nanoparticles (LNP-poly(I:C)). Moreover, LNG-poly(I:C) induced more potent CD8+ T cell immunity and stronger therapeutic effects than LNP-poly(I:C). The combination of LNG-poly(I:C) and PD-L1 targeting led to regression of established lung metastases. Due to the ease of manufacturing and the high biocompatibility of LNG, pulmonary delivery of LNG may be broadly applicable to the treatment of different lung tumors and may spur the development of innovative strategies for cancer immunotherapy.

Hepatocellular carcinoma (HCC) is an aggressive disease that occurs predominantly in men. Estrogen elicits protective effects against HCC development. Elucidation of the estrogen-regulated biological processes that suppress HCC could lead to improved prevention and treatment strategies. Here, we performed transcriptomic analyses on mouse and human liver cancer and identified LCAT as the most highly estrogen-upregulated gene and a biomarker of favorable prognosis. LCAT upregulation inhibited HCC in vitro and in vivo and mediated estrogen-induced suppression of HCC in an ESR1-dependent manner. LCAT facilitated high-density lipoprotein cholesterol (HDL-C) production and uptake via the LDLR and SCARB1 pathways. Consistently, high HDL-C levels corresponded to a favorable prognosis in HCC patients. The enhanced HDL-C absorption induced by LCAT impaired SREBP2 maturation, which ultimately suppressed cholesterol biosynthesis and dampened HCC cell proliferation. HDL-C alone inhibited HCC growth comparably to the cholesterol-lowering drug lovastatin, and SREBF2 overexpression abolished the inhibitory activity of LCAT. Clinical observations and cross-analyses of multiple databases confirmed the correlation of elevated LCAT and HDL-C levels to reduced cholesterol synthesis and improved HCC patient prognosis. Furthermore, LCAT deficiency mimicked whereas LCAT overexpression abrogated the tumor growth promoting effects of ovariectomy in HCC-bearing female mice. Most importantly, HDL-C and LCAT delayed the development of subcutaneous tumors in nude mice, and HDL-C synergized with lenvatinib to eradicate orthotopic liver tumors. Collectively, this study reveals that estrogen upregulates LCAT to maintain cholesterol homeostasis and dampen hepatocarcinogenesis. LCAT and HDL-C represent potential prognostic and therapeutic biomarkers for targeting cholesterol homeostasis as a strategy for treating HCC.

Abstract Background: Obesity is associated with higher risk of breast cancer (BC)-related death, all-cause mortality, and recurrence. While used to estimate body fat, body mass index (BMI) is insensitive to body fat distribution and lean muscle mass. We hypothesized that BMI categories (BMI 18.5-24.9 [healthy], 25-29.9 [overweight], and ≥30 kg/m2 [obesity]) would not correlate with CT-derived body composition. Methods: We retrospectively identified 180 patients diagnosed with new or recurrent Stage I-IV BC who presented to Johns Hopkins from 2015-2018 and were part of an institutional database. We extracted demographics, diagnosis date, cancer characteristics, BMI, and CT abdomen pelvis scans from the medical record. Baseline BMI was defined as the closest measurement to diagnosis between 1 year prior and 1 month after diagnosis. Baseline CT was within 6 months of and closest to the baseline BMI date. Fully automated deep-learning algorithms identified L1 and performed muscle and adipose tissue segmentation and quantification. Body composition data included: cross-sectional areas (CSA) of subcutaneous, visceral, and total adipose tissue; the mean, median, and standard deviation of the average of the muscle attenuation (density); and the body wall musculature CSA. ANOVA tests assessed associations between body composition, BMI, and menopausal status. Results: Among 180 patients, 136 (76%) had early-stage BC, and 44 (24%) had metastatic BC. Most patients were women (98%) and non-Hispanic (94%). Patients identified as White (57%), Black (29%), Asian (6%), and Other Race (8%). Sixty percent were post-menopausal. Hormone receptor-positive, HER2-positive, and triple negative subtypes were 56%, 19% and 22%, respectively. At diagnosis, 24% had a healthy BMI while 36% and 41% had overweight and obesity, respectively. All body composition measures were significantly associated with BMI (Table 1). As BMI increased, the average body wall musculature and subcutaneous, visceral, and total adipose tissue CSAs increased while the average muscle attenuation decreased. Specifically, the average visceral adipose tissue CSAs were 2.7 and 4.0-fold larger for overweight and obesity compared to that of the healthy BMI group, respectively, while their average muscle attenuations were 62.4% and 40.6% of the healthy BMI group. Postmenopausal women had 1.5-fold higher visceral adipose tissue CSA and 44.7% less average muscle attenuation compared to premenopausal women. Conclusion: Body composition correlates with all BMI categories in primarily white, non-Hispanic women with breast cancer. As BMI increases, body composition changes reliably, with increased subcutaneous and visceral adipose tissue, increased muscle area, and decreased muscle density. Muscle density, not size/area, is more associated with muscle performance, which can relate to functional status and outcomes. Despite validity concerns, BMI is an accessible and economical surrogate for body adiposity in patients with breast cancer. Postmenopausal status was also associated with greater visceral and total adiposity and lower muscle density. Further investigation is needed to confirm these findings. Table 1: Correlation between body composition, BMI, and menopausal status in patients with breast cancer All measures of body composition were significantly associated with BMI. Visceral adipose tissue increased and muscle density decreased as BMI increased and with post-menopausal status. Citation Format: Terrence Tsou, Tingchang Wang, Amanda Blackford, Ronald Summers, Vered Stearns, Jennifer Sheng. Body composition correlates with BMI and menopausal status in patients with breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-11-09.

Abstract Background: Massive fungating breast cancers (MFBCs), which comprise exudative, foul-smelling tumors, the size of a child’s head or involve extensive ulceration are classified as locally advanced breast cancers. The National Comprehensive Cancer Network guidelines recommend preoperative neoadjuvant chemotherapy in patients with locally advanced breast cancer without distant metastases. However, limited evidence is available regarding the successful treatment of fungating breast cancers. In clinical practice, many fungating breast cancer cases are treated as stage IV tumors. This study aimed to investigate whether multimodal therapy, including surgery for giant breast cancers, can improve survival rates and whether the survival rates are comparable to those of other stage III breast cancers. Subjects and methods: Out of 1480 cases of primary breast cancer diagnosed at our hospital between June 2012 and March 2020, 123 patients with clinical stages IIIA–IIIC were chosen for analysis. Furthermore, 29 of these patients with MFBC were selected. During multimodal therapy for selected patients with fungating breast cancer, difficulties were encountered in some patients with single-stage skin suturing and closure after standard neoadjuvant chemotherapy. These patients underwent surgery with an additional skin flap or skin grafting. Furthermore, radiation therapy was added as needed. We compared the survival rates of these patients with those of patients who received drug therapy only according to the therapeutic protocol for Stage IV. Survival rates of patients with MFBC and other stage III breast cancers were also compared. Statistical analyses were conducted using the Kaplan–Meier curve and the log-rank test. Results: Of the 29 patients with MFBC, 22 (75.9%) and seven (24.1%) were treated with multimodal and palliative therapies, respectively. The median age and follow-up period of the multimodal therapy group were 62 (range: 43–94) years and 67 (range, 32–131) months, respectively. The corresponding values for the palliative therapy group were 79 (range: 42–88) years and 31 (range: 2–96) months. All patients in the palliative treatment group were hormone receptor positive and HER2 negative, and treatment with hormone therapy was initiated. The 7-year survival rate was significantly higher in the multimodal therapy group than in the palliative treatment group (92.3% vs. 68.6%, p = 0.0317). Furthermore, the 5-year distant recurrence-free rate was higher in the multimodal treatment group (79.3% vs. 63.5%). Local recurrence did not occur in any patient in the multimodal treatment group. There was no significant difference in the 7-year survival rates between patients with MFBC and those with other stage III breast cancers (86.8% vs. 75.3%; p = 0.239). Triple-negative breast cancer accounted for 20.8% (20 96) of the other stage III breast cancers, but none of these were MFBCs. Conclusions: Clinically, MFBCs are often palliatively treated as stage IV tumors, even in the absence of distant metastasis. However, the survival rate was significantly higher in the multimodal therapy group than in the palliative treatment group. Furthermore, the survival and distant recurrence-free rates for MFBC were higher than those for other stage III breast cancers. Therefore, patients with MFBCs should be actively treated using curative treatments. Citation Format: Tamaki Tamanuki, Maki Namura, Tomoyoshi Aoyagi, Haruhito Sakata, Mika Iwai, Shinichirou Shimizu, Hiroshi Matsuzaki. Assessing the effect of multimodal therapy in massive fungating breast cancers without distant metastasis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-26-01.

Abstract A fibrotic focus (FF) is a scar-like region that forms in the center of carcinoma due to excessive tumor stroma formation. In our earlier studies, we found FF as an independent poor prognostic factor for breast cancers. However, the underlying mechanisms of how the presence of FF contributed to a poor prognosis are still unknown. To improve our understanding of FF in breast cancer, we have investigated genes associated with FF and their impacts on breast cancer prognosis. Analysis of data from The Cancer Genome Atlas Program (TCGA) indicated that Bone Morphogenetic Protein 8A (BMP8A) was significantly upregulated in breast cancers with FF. To further validate this finding, immunohistochemistry (IHC) on BMP8A was performed on a local cohort of breast cancers (N= 900). Of these samples, 147 (16.3%) were found to be BMP8A positive, and there was a significant association between the presence of FF and high expression of BMP8A (p-value=0.026). We also found that high expression of BMP8A was positively correlated with apocrine phenotype, necrosis, increased Ki67 expression, and triple-negative breast cancer subtype; and most of these are poor prognostic factors for breast cancer. Kaplan-Meier survival estimate analysis revealed that high expression of BMP8A was significantly associated with poor overall survival (p-value=0.035). Gene ontology (GO) analysis showed that "Extracellular matrix organization" was the prominent biological process associated with BMP8A. KEGG pathway analysis suggested that BMP8A was significantly related to "Protein digestion and absorption", "ECM-receptor interaction", and "Focal adhesion" pathways, further validating its correlation with stromal FF. In cellular studies using MCF7 and MDA-MB-231 cell lines, overexpression of BMP8A was found to enhance breast cancer cell proliferation, invasion, and migration. Similar tumor-promoting phenotypes were exhibited when stimulating the cell lines with recombinant human BMP8A. In conclusion, our findings suggested that BMP8A was associated with FF in breast cancer, contributing at least partly to the aggressive features of these cancers. Our findings also provided new insights into the precision treatment and diagnosis of breast cancer. Further exploration of BMP8A as a therapeutic target and prognostic factor may contribute to improved management of breast cancer patients. Citation Format: Canbin Fang, Julia Y. S. Tsang, Gary M.K. Tse. Mechanistic Analysis and Potential Therapeutic Implications in Breast Cancer with Fibrotic Focus [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-13-12.