Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the bone marrow. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-sequencing to characterize immune and tumor cell alterations in neuroblastoma bone marrow metastases by comparative analysis with patients without metastases. Our results revealed remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the bone marrow metastatic niche we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of regulatory T cells and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and identified prognostic markers in malignant cells that are associated with worse clinical outcomes in three independent neuroblastoma cohorts. Our results provide insights into the cellular, compositional and transcriptional shifts underlying neuroblastoma bone marrow metastases contributing to the development of new therapeutic strategies.
Shenglin Mei, Adele M. Alchahin, Bethel Tesfai Embaie, Ioana Maria Gavriliuc, Bronte Manouk Verhoeven, Ting Zhao, Xiangyun Li, Nathan Elias Jeffries, Adena Pepich, Hirak Sarkar, Thale Kristin Olsen, Malin Wickström, Jakob Stenman, Oscar Reina-Bedoya, Peter V. Kharchenko, Philip J. Saylor, John Inge Johnsen, David B. Sykes, Per Kogner, Ninib Baryawno
Immune checkpoint inhibitors (ICIs) are indicated for a diverse range of cancer types, and characterizing the tumor immune microenvironment is critical for optimizing therapeutic strategies, including ICIs. T cell infiltration and activation status in the tumor microenvironment greatly affects the efficacy of ICIs. Here, we show that semaphorin 6D (Sema6D) forward signaling, which is reportedly involved in coordinating the orientation of cell development and migration as a guidance factor, impaired the infiltration and activation of tumor-specific CD8+ T cells in murine oral tumors. Sema6D expressed by nonhematopoietic cells was responsible for this phenotype. Plexin-A4, a receptor for Sema6D, inhibited T cell infiltration and partially suppressed CD8+ T cell activation and proliferation induced by Sema6D stimulation. Moreover, mouse oral tumors, which are resistant to PD-1–blocking treatment in wild-type mice, showed a response to the treatment in Sema6d-KO mice. Finally, analyses of public data sets of human head and neck squamous cell carcinoma, pan-cancer cohorts, and a retrospective cohort study showed that SEMA6D was mainly expressed by nonhematopoietic cells such as cancer cells, and SEMA6D expression was significantly negatively correlated with CD8A, PDCD1, IFNG, and GZMB expression. Thus, targeting Sema6D forward signaling is a promising option for increasing ICI efficacy.
Takashi Hirai, Yujiro Naito, Shohei Koyama, Yoshimitsu Nakanishi, Kentaro Masuhiro, Mayuko Izumi, Tomoki Kuge, Maiko Naito, Yumiko Mizuno, Yuta Yamaguchi, Sujin Kang, Moto Yaga, Yu Futami, Satoshi Nojima, Masayuki Nishide, Takayoshi Morita, Yasuhiro Kato, Takeshi Tsuda, Norihiko Takemoto, Yumi Kinugasa-Katayama, Taiki Aoshi, Jordan Kelly Villa, Kazuo Yamashita, Tomohiro Enokida, Yuta Hoshi, Kazuto Matsuura, Makoto Tahara, Hyota Takamatsu, Yoshito Takeda, Hidenori Inohara, Atsushi Kumanogoh
The gut and local esophageal microbiome progressively shift from healthy commensal bacteria to inflammatory-linked pathogenic bacteria in patients with gastroesophageal reflux disease, Barrett’s esophagus and esophageal adenocarcinoma (EAC). However, mechanisms by which microbial communities and metabolites contribute to reflux-driven EAC remain incompletely understood and challenging to target. Herein, we utilized a rat reflux-induced EAC model to investigate targeting the gut microbiome-esophageal metabolome axis with cranberry proanthocyanidins (C-PAC) to inhibit EAC progression. Sprague Dawley rats, with or without reflux-induction received water or C-PAC ad libitum (700 µg/rat/day) for 25 or 40 weeks. C-PAC exerted prebiotic activity abrogating reflux-induced dysbiosis, and mitigating bile acid metabolism and transport, culminating in significant inhibition of EAC through TLR/NF-κB/TP53 signaling cascades. At the species level, C-PAC mitigated reflux-induced pathogenic bacteria (Streptococcus parasanguinis, Escherichia coli, and Proteus mirabilis). C-PAC specifically reversed reflux-induced bacterial, inflammatory and immune-implicated proteins and genes including Ccl4, Cd14, Crp, Cxcl1, Il6, Il1β, Lbp, Lcn2, Myd88, Nfkb1, Tlr2, and Tlr4 aligning with changes in human EAC progression, as confirmed through public databases. C-PAC is a safe promising dietary constituent that may be utilized alone or potentially as an adjuvant to current therapies to prevent EAC progression through ameliorating reflux-induced dysbiosis, inflammation and cellular damage.
Katherine M. Weh, Connor L. Howard, Yun Zhang, Bridget A. Tripp, Jennifer L. Clarke, Amy B. Howell, Joel H. Rubenstein, Julian A. Abrams, Maria Westerhoff, Laura A. Kresty
High grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to biobehavioral sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines are able to survive in ultra-low attachment (ULA) culture in a beta-adrenergic receptor (β-AR)-dependent manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE can be abrogated using the beta-adrenergic receptor blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony stimulating factor 2 (CSF2). These findings provide mechanistic insight and identify targets that may be regulated by ovarian-derived NE in early HGSC.
Hunter D. Reavis, Stefan M. Gysler, Grace B. McKenney, Matthew Knarr, Hannah J. Lusk, Priyanka Rawat, Hannah S. Rendulich, Marilyn A. Mitchell, Dara S. Berger, Jamie S. Moon, Suyeon Ryu, Monica Mainigi, Marcin P. Iwanicki, Dave S.B. Hoon, Laura M. Sanchez, Ronny Drapkin
Metastatic breast cancer (mBC) tissue in bone was systematically profiled to define the composition of the tumor microenvironment. Gene expression identified a high myeloid signature of patients with improved survival outcomes. Bone metastases were profiled by spatial proteomics to examine myeloid populations within the stroma that correlated with macrophage functions. Single-cell spatial analysis uncovered macrophage activation in the stroma of mBC bone lesions. Matched BC patient samples of primary breast tumor and bone metastasis tissues were compared for gene expression in the bone, where bone morphogenetic protein 2 (BMP2) was most significantly upregulated. Immune cell changes from breast to bone demonstrated a loss of lymphoid cells but a consistent population of macrophages. BMP-activated macrophages were increased uniquely in bone. Bone marrow–derived macrophage activation coupled with BMP inhibition increased inflammatory responses. Using experimental mouse models of mBC bone metastasis and trained immunity, we found that BMP inhibition restricts progression of metastases early in the macrophage activation state but not after tumors were established in the bone. This study revealed unique myeloid BMP activation states that are distinctly integrated with bone metastases.
Claire L. Ihle, Desiree M. Straign, Johana A. Canari, Kathleen C. Torkko, Kathryn L. Zolman, Elizabeth E. Smith, Philip Owens
Dissemination within the peritoneal cavity is a main determinant of poor patient outcomes from high-grade serous carcinomas (HGSCs). The dissemination process is poorly understood from a cancer evolutionary perspective. We reconstructed the evolutionary trajectories across a median of five tumor sites and regions from each of 23 patients (n=108 samples) based on deep whole-exome sequencing. Polyclonal cancer origin was detected in one patient. Ovarian tumors had more complex subclonal architectures than other intra-peritoneal tumors in each patient, which indicated that tumors developed earlier in the ovaries. Three common modes of dissemination were identified, including monoclonal (27%) or polyclonal dissemination of monophyletic (linear; 50%) or polyphyletic (branched; 23%) subclones. Mutation profiles of initial or disseminated clones varied greatly among cancers, but recurrent mutations were found in seven cancer-critical genes, such as TP53, BRCA1, BRCA2, DNMT3A, and in the PI3K/AKT1 pathway. Disseminated clones developed late in the evolutionary trajectory models of most cancers, in particular in cancers with DNA damage repair deficiency. Polyclonal dissemination was predicted to occur predominantly as a single and rapid wave, but chemotherapy exposure was associated with higher genomic diversity of disseminated clones. In conclusion, we described three common evolutionary dissemination modes across HGSCs and proposed factors associated with dissemination diversity.
Anita Sveen, Bjarne Johannessen, Solveig M.K. Klokkerud, Sigrid M. Kraggerud, Leonardo A. Meza-Zepeda, Merete Bjørnslett, Katharina Bischof, Ola Myklebost, Kjetil Taskén, Rolf I. Skotheim, Anne Dørum, Ben Davidson, Ragnhild A. Lothe
Heterologous polyclonal antibodies (pAb) were shown to possess oncolytic properties a century ago with reported clinical responses. More recent pre-clinical models confirm pAb efficacy though their ability to tackle complex target antigens, which reduces susceptibility to tumor escape. Owing to the recent availability of glyco-humanized pAbs with acceptable clinical safety profile, we revisited use of pAbs in oncology and highlighted their therapeutic potential against multiple cancer types. Murine anti-tumor pAbs were generated after repeated immunization of rabbits with murine tumor cell lines from hepatocarcinoma, melanoma, and colorectal cancers. Anti-tumor pAbs recognized and showed cytotoxicity against their targets without cross-reactivity with healthy tissues. In vivo, pAbs are effective alone, moreover, these pAbs synergize with, immune checkpoints inhibitors like anti-PDL-1 in several cancer models. They elicited an anti-tumor host immune response and prevented metastases. The anticancer activity of pAbs was also confirmed in xenografted NMRI nude mice using glyco-humanized pAbs (GH-pAbs) produced by repeated immunization of pigs with human tumor cell lines. In conclusion, the availability of bioengineered GH-pAb allows to revisit passive immunotherapy with oncolytic pAb to fight against solid tumor and cancer metastasis.
Carine Ciron, Pierre Morice, Juliette Rousse, Patrice Roy, Pierre-Joseph Royer, Olivier Gauthier, Sophie Brouard, Odile Duvaux, Firas Bassissi, Bernard Vanhove
IL-12 is a potent cytokine that can promote innate and adaptive anticancer immunity, but its clinical development has been limited by toxicity when delivered systemically. Intratumoral (i.t.) administration can expand the therapeutic window of IL-12 and other cytokines but is in turn limited by rapid drug clearance from the tumor, which reduces efficacy, necessitates frequent administration, and increases systemic accumulation. To address these limitations, we developed an anchored IL-12 designated ANK-101, composed of an engineered IL-12 variant that forms a stable complex with the FDA-approved vaccine adjuvant aluminum hydroxide (Alhydrogel). Following i.t. administration of murine ANK-101 (mANK-101) in early intervention syngeneic mouse tumors, the complex formed a depot that was locally retained for weeks as measured by IVIS or SPECT/CT imaging, while unanchored protein injected i.t. was cleared within hours. One or 2 i.t. injections of mANK-101 induced single-agent antitumor activity across a diverse range of syngeneic tumors, including models resistant to checkpoint blockade at doses where unanchored IL-12 had no efficacy. Local treatment with mANK-101 further induced regressions of noninjected lesions, especially when combined with systemic checkpoint blockade. Antitumor activity was associated with remodeling of the tumor microenvironment, including prolonged IFN-γ and chemokine expression, recruitment and activation of T and NK cells, M1 myeloid cell skewing, and increased antigen processing and presentation. Subcutaneous administration of ANK-101 in cynomolgus macaques was well tolerated. Together, these data demonstrate that ANK-101 has an enhanced efficacy and safety profile and warrants future clinical development.
Sailaja Battula, Gregory Papastoitsis, Howard L. Kaufman, K. Dane Wittrup, Michael M. Schmidt
Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I– (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell–specific neoantigens for personalized cancer vaccine modalities.
Amanda L. Huff, Gabriella Longway, Jacob T. Mitchell, Lalitya Andaloori, Emily Davis-Marcisak, Fangluo Chen, Melissa R. Lyman, Rulin Wang, Jocelyn Mathew, Benjamin Barrett, Sabahat Rahman, James Leatherman, Mark Yarchoan, Nilofer S. Azad, Srinivasan Yegnasubramanian, Luciane T. Kagohara, Elana J. Fertig, Elizabeth M. Jaffee, Todd D. Armstrong, Neeha Zaidi
The use of patient-derived organoids (PDOs) to characterize therapeutic sensitivity and resistance is a promising precision medicine approach, and its potential to inform clinical decisions is now being tested in several large multi-institutional clinical trials. PDOs are cultivated in extracellular matrix from basement membrane extracts (BMEs) that are most commonly acquired commercially. Each clinical site utilizes distinct BME lots and may be restricted due to the availability of commercial BME sources. However, the impact of different sources of BMEs on organoid drug response is unknown. Here, we tested the impact of BME source on proliferation, drug response, and gene expression in mouse and human pancreatic ductal adenocarcinoma (PDA) organoids. Both human and mouse organoids displayed increased proliferation in Matrigel (Corning) compared to Cultrex (RnD) and UltiMatrix (RnD). However, we observed no substantial impact on drug response when organoids were cultured in Matrigel, Cultrex, or UltiMatrix. We also did not observe major shifts in gene expression across the different BME sources, and PDOs maintained their Classical or Basal-like designation. Overall, we find that BME source (Matrigel, Cultrex, UltiMatrix) does not shift PDO dose-response curves and drug testing results, indicating that PDO pharmacotyping is a robust approach for precision medicine.
Jan C. Lumibao, Shira R. Okhovat, Kristina L. Peck, Xiaoxue Lin, Kathryn Lande, Shira Yomtoubian, Isabella Ng, Herve Tiriac, Andrew M. Lowy, Jingjing Zou, Dannielle D. Engle
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