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Although macrophages contribute to cancer cell dissemination, immune evasion, and metastatic outgrowth, they have also been reported to coordinate tumor-specific immune responses. We therefore hypothesized that macrophage polarization could be modulated therapeutically to prevent metastasis. Here, we show that macrophages respond to β-glucan (odetiglucan) treatment by inhibiting liver metastasis. β-glucan activated liver-resident macrophages (Kupffer cells), suppressed cancer cell proliferation, and invoked productive T cell-mediated responses against liver metastasis in pancreatic cancer mouse models. Although excluded from metastatic lesions, Kupffer cells were critical for the anti-metastatic activity of β-glucan, which also required T cells. Furthermore, β-glucan drove T cell activation and macrophage re-polarization in liver metastases in mice and humans and sensitized metastatic lesions to anti-PD1 therapy. These findings demonstrate the significance of macrophage function in metastasis and identify Kupffer cells as a potential therapeutic target against pancreatic cancer metastasis to the liver. The liver is the most common site of metastasis for pancreatic ductal adenocarcinoma (PDAC). Here, the authors demonstrate that β-glucan, a microbial component associated with trained immunity, activates liver-resident macrophages (Kupffer cells) and prevents PDAC metastasis to the liver

The efficacy of live attenuated bacterial vectors is dependent upon the fine-tuning of a strain’s immunogenicity and its virulence. Strains are often engineered to deliver heterologous antigens, but soluble expression of recombinant proteins can be troublesome. Therefore, secretion systems or chaperone proteins are routinely used to assist in attaining high levels of functional, soluble protein production. However, the effects of chaperone expression on the virulence of attenuated bacterial vectors have not been previously reported. In anticipation of utilizing periplasmic chaperone proteins to facilitate soluble production of immunomodulatory proteins in an attenuated strain of Salmonella Typhimurium, the production of the chaperones was tested for their effect on both culture growth and bacterial persistence in mouse tissues. Although no effect on growth of the bacteria was observed in vitro, the increased expression of the periplasmic chaperones resulted in over-attenuation of the Salmonella in vivo.

Abstract Background HC-7366 is a novel, orally administered, highly selective and potent activator of general control nonderepressible 2 (GCN2) kinase, a core regulator of metabolic stress through activation of the integrated stress response (ISR). Activation of GCN2 promotes cell survival, whereas prolonged activation induces apoptosis. GCN2 activation also suppresses general protein synthesis and induces cell cycle arrest, thereby preventing cell growth during nutrient scarcity. Additionally, HC-7366 decreases HIF expression and inhibits glycolysis, oxidative phosphorylation, and TCA cycle function. HC-7366 also inhibits HIF expression in immunosuppressive myeloid cells, including macrophages. These effects of HC-7366 on metabolism, HIF signaling, and immune suppression suggest therapeutic benefit in ccRCC with clear rationale for combinations with HIF2a antagonists and immune checkpoint inhibitors. HC-7366 (0.5-1 mg/kg), combined with belzutifan (1 mg/kg), exhibited combination benefit in HIF-2 dependent A-498 and 786-O RCC xenografts, yielding 90% tumor growth inhibition and a three-fold increase in complete responses, respectively. Additionally, HC-7366 drives significant monotherapy antitumor activity in PDX models that demonstrated belzutifan resistance. Mechanism of action studies have identified several pathway engagement and potential efficacy biomarkers (Stokes, et al., 2024). Trial design/schema This multicenter, open-label phase 1b study will identify the Maximum Tolerated Dose (MTD) and/or the Recommended Phase 2 Dose (RP2D) of HC-7366 in combination with fixed-dose belzutifan, 120 mg po qd, in patients with advanced or metastatic RCC with renal cell histology irrespective of VHL gene mutation. Monotherapy HC-7366 will be evaluated in parallel. The monotherapy arm, HC-7366, 60 mg p.o. qd, includes patients who have relapsed after 1 to 4 prior lines of standard of care that may include belzutifan or another HIF-2α inhibitor. The combination arm includes patients who have received 1 to 3 prior lines of standard of care and are belzutifan or HIF-2α naïve. HC-7366 dose escalation arm evaluates combination fixed dose belzutifan, 120 mg po qd plus HC-7366 at 20, 40, or 60 mg po qd. Enrollment will start with the HC-7366 monotherapy arm. Subsequently, patients will begin to enroll in the combination therapy HC-7366 dose escalation arm, while the monotherapy arm will continue to enroll. Expansion will evaluate the combination of fixed dose belzutifan with two doses of HC-7366 selected from escalation. Assessments include safety, PK, and anti-tumor activity. The study will enroll up to 80 patients at US study sites. Significance and vision Combination HC-7366 plus belzutifan, supported by preclinical evidence, is being studied to assess antitumor activity in the RCC relapsed setting. Determining safety and evaluation of dose are foundational to this study. Reference Stokes M, Tameire F, Wojnarowicz P, et al. HC-7366, a potent GCN2 activator, complements belzutifan, a HIF-2⍺ antagonist, by providing combination benefit in belzutifan-sensitive models and monotherapy activity in belzutifan-resistant models. Meeting of the American Association for Cancer Research; 2024 Apr 5-10; San Diego (CA); AACR; 2024. Abstract 4615. This study is in collaboration with Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA

BackgroundProtein kinase R-like endoplasmic reticulum kinase (PERK) is part of the unfolded protein response that facilitates cellular adaptation to ER stress. PERK is activated in cancer cells by accumulation of misfolded proteins in the ER, enabling adaptation and survival. PERK signaling has also recently been implicated in maintaining immunosuppressive functions of myeloid-derived suppressor cells (MDSCs) through inhibition of a type 1 interferon response1 and macrophages through metabolic and epigenetic modification.2 We are developing HC-5404, a highly selective and potent first-in-class, first-in-human PERK inhibitor that is currently in a phase 1 trial for solid tumors (NCT04834778). HC-5404 has demonstrated single agent and combinatorial efficacy in multiple solid tumor xenograft models. In this study, we sought to investigate the immunomodulatory effects of HC-5404 by evaluating efficacy and correlative immune effects of HC-5404 combined with an anti-murine-PD-1 immune checkpoint inhibitor (ICI) antibody.MethodsC57BL/6 mice were subcutaneously inoculated with syngeneic MB49 bladder cancer cells, and treatment started on day 8 post cell inoculation. A group of animals (n=10/group) received either vehicle, HC-5404 (PO, BID), anti-PD-1 antibody RMP1-14 (IP, every 3 days), or the combination of both. At various timepoints, flow cytometry was performed on blood or single cell suspensions from tumors or lymph nodes (n=6) of treated mice. MDSCs derived from human cord blood or mouse bone marrow were co-cultured with purified T-cells in the presence of HC-5404 in vitro, and proliferation was evaluated.ResultsHC-5404 treatment alone showed only a modest anti-tumor effect (32% TGI), the addition of HC-5404 to aPD-1 provided combination antitumor benefits (75% TGI) and significantly improved the effects of aPD-1 alone (53% TGI). HC-5404 + aPD-1 efficacy was correlated with increased expression of type 1 interferon receptor (IFNAR1) and increased surface calreticulin on tumor cells. Additionally, IFNAR1 was also significantly increased on PMN-MDSCs and tumor-associated macrophages (TAM). TAMs also showed increased PD-L1 with combination treatment. Additionally, combination treatment increased the frequency of CD8 T-cells in the tumor along with increased expression of activation marker CD69 on T-cells in the tumor draining lymph node. Notably, the effect of HC-5404 on IFNAR1 was also detected on monocytes in peripheral blood, demonstrating surface expression of IFNAR1 as a potential biomarker for HC-5404 activity. MDSCs also showed a reduced inhibition of T-cells in the presence of HC-5404 in vitro.ConclusionsCollectively, these data demonstrate the efficacious and immuno-stimulatory effects of HC-5404 coadministered with anti-PD1 mAb and outline its potential application in ICI-treated cancers.ReferencesMohamed E, et al. The unfolded protein response mediator perk governs myeloid cell-driven immunosuppression in tumors through inhibition of STING signaling. Immunity. 2020;52(4): 668–682. e667.Raines LN, et al. PERK is a critical metabolic hub for immunosuppressive function in macrophages. Nat Immunol 2022;23(3): 431–445.Ethics ApprovalAll in vivo experimental procedures were done in accordance with the NIH Guide for Care and Use of Animals and were approved by the Institutional Animal Care and Use Committee of University of Minnesota. IACUC protocol 2009A38458

Interest in cancer immunotherapy has grown in recent years due to its potential for significant and durable therapeutic responses. Immune checkpoint blockade has emerged as an immunotherapy as a single agent but has even greater appeal when it is used in combination with other immunostimulatory approaches. However, the dosing of checkpoint blockade and its combinatorial use with other immunotherapies has been limited by systemic immune-related adverse side effects. One way to overcome these adverse effects is to deliver the therapeutic agents specifically to the tumor microenvironment. Salmonella enterica Typhimurium (S. Typhimurium) has been studied for cancer therapy due to its genetic manipulability and tumor-targeting propensity, and in this thesis, the potential of S. Typhimurium as a tumor-targeting immunotherapy vector was investigated. Functional antagonistic single chain antibodies (scFvs) against the immune checkpoints CTLA-4 and PD-L1 were isolated from an immunized chicken library and engineered for secretion from S. Typhimurium. The inherent anti-tumor properties and tumor-targeting capability of S. Typhimurium were then tested in transplanted primary and metastatic tumor models as well as a genetically engineered autochthonous BALB-neuT breast cancer model. In each of these models, S. Typhimurium demonstrated native anti-tumor efficacy; however the bacteria did not adequately colonize the autochthonous tumors of the BALB-neuT model. Disruption of tumor vasculature by treating BALB-neuT mice with a vasculature disrupting agent (VDA) improved the colonization of autochthonous tumors over 1000-fold to levels similar to those observed for transplanted tumors. Subsequent comparison of the tumor targeting capability and efficacy of S. Typhimurium engineered to secrete the antagonistic αPD-L1 (scFv) versus a control strain showed that secretion of the scFv may further improve the colonization of autochthonous tumors, leading to a greater reduction in tumor burden of treated mice. These findings provide a proof of principle for the expression and delivery of functional immunotherapeutic single chain antibodies using S. Typhimurium, demonstrate S. Typhimurium’s native tumoricidal activity independent of tumor-targeting, illustrate the importance of clinically representative tumor models when studying bacterial cancer therapy, and demonstrate the potential of VDA treatment to improve bacterial tumor-targeting. Collectively, this work illustrates S. Typhimurium’s promise as a tumor-targeting immunotherapy vector.

Species’ ranges are dynamic, changing through range shifts, contractions, and expansions. Individuals at the edge of a species’ shifting range often possess morphological traits that increase movement capacity, that are not observed in individuals farther back within the species’ range. Although morphological traits that increase in proportion toward the range edge may differ between the sexes, such sex differences are rarely studied. Here, we test the hypotheses that body size and condition increase with proximity to an expanding range edge in the flightless ground beetle, Carabus hortensis, and that these trait changes differ between the sexes. Male, but not female, body size increased with proximity to the range edge. Body size was positively correlated with male front and mid tibia length and to female hind tibia length, indicating that body size is indicative of movement capacity in both sexes. Body condition (relative to body size) decreased with increasing population density in males but not females. Population density was lowest at the range edge. Our results indicate that sex is an important factor influencing patterns in trait distribution across species’ ranges, and future studies should investigate changes in morphological traits across expanding range margins separately for males and females. We discuss the implications for sex differences in resource allocation and reproductive rates for trait differentiation across species’ shifting ranges. The distribution of morphological traits may change across species' shifting or expanding ranges. Here, we show that the differentiation of such traits is sex‐specific in the ground beetle species Carabus hortensis: Male, but not female, body size increased with proximity to the range edge.

Individual differences in movement patterns are increasingly recognised as important within ecology. In the laboratory, they are, however, often quantified through relatively coarse measures. We describe a low‐cost trackball system incorporating a 3D‐printed holder, commercially available polystyrene ball and implemented with open‐source software. We used this system to record six parameters of walking behaviour (e.g., translational velocity, total rotation) and tested the hypothesis that these walking parameters are repeatable across individuals. We tested 30 ground beetle individuals, Carabus problematicus, each in two trackball trials 1 week apart. Individuals were repeatable and differed consistently in several movement parameters (including distance walked, translational velocity and path straightness) but not in others (e.g., total rotation and sinuosity). Trackballs allow quantification of walking parameters for a wide range of animals, enabling identification of individual differences in specific aspects of walking, both fine‐scale and long‐distance aspects of movement. In doing so, trackballs offer novel insights into behavioural ecology, including consistent animal personality differences. We describe a low‐cost trackball with automated movement capture for use in behavioural ecology. Repeated testing of ground beetles Carabus problematicus showed consistent behavioural differences between individuals in movement parameters. Trackballs offer novel insights into behavioural ecology, including consistent animal personality differences.

Individuals within the same species often differ in their metabolic rates, which may covary with behavioural traits (such as exploration), that are consistent across time and/or contexts, and morphological traits. Yet, despite the frequent occurrence of sexual dimorphisms in morphology and behaviour, few studies have assessed whether and how sexes differ in metabolic trait covariances. We investigated sex-specific relationships among resting or active metabolic rate (RMR and AMR, respectively) with exploratory behaviour, measured independently of metabolic rate in a novel environment, body size and body mass, in ground beetles. RMR, AMR and exploratory behaviour were repeatable among individuals across time, except for male RMR which was unrepeatable. Female RMR neither correlated with exploratory behaviour nor body size/body mass. In contrast, AMR was correlated with both body size and exploratory behaviour. Males with larger body sizes had higher AMR, whereas females with larger body sizes had lower AMR. Both male and female AMR were significantly related to exploratory behaviour, though the relationships between AMR and exploration were body mass-dependent in males and temperature-dependent in females. Differences between sexes exist in the covariances between metabolic rate, body size and exploratory behaviour. This suggests that selection acts differently on males and females to produce these trait covariances with potentially important consequences for individual fitness.