Fresh Human Tumor Research Articles

Claudin 7 suppresses invasion and metastasis through repression of a smooth muscle actin program.

Metastasis initiates when cancer cells escape from the primary tumor, which requires changes to intercellular junctions. Claudins are transmembrane proteins that form the tight junction, and their expression is reduced in aggressive breast tumors. However, claudins' roles during breast cancer metastasis remain unclear. We used gain- and loss-of-function genetics in organoids isolated from murine breast cancer models to establish that Cldn7 suppresses invasion and metastasis. Transcriptomic analysis revealed that Cldn7 knockdown induced smooth muscle actin (SMA)-related genes and a broader mesenchymal phenotype. We validated our results in human cell lines, fresh human tumor tissue, bulk RNA-seq, and public single-cell RNA-seq data. We consistently observed an inverse relationship between Cldn7 expression and expression of SMA-related genes. Furthermore, knockdown and overexpression of SMA-related genes demonstrated that they promote breast cancer invasion. Our data reveal that Cldn7 suppresses breast cancer invasion and metastasis through negative regulation of SMA-related and mesenchymal gene expression.

Injectable and Sprayable Fluorescent Nanoprobe for Rapid Real-Time Detection of Human Colorectal Tumors.

The development of minimally invasive surgery has greatly advanced precision tumor surgery, but sometime suffers from restricted visualization of the surgical field, especially during the removal of abdominal tumors. A 3-D inspection of tumors could be achieved by intravenously injecting tumor-selective fluorescent probes, whereas most of which are unable to instantly distinguish tumors via in situ spraying, which is urgently needed in the process of surgery in a convenient manner. In this study, this work has designed an injectable and sprayable fluorescent nanoprobe, termed Poly-g-BAT, to realize rapid tumor imaging in freshly dissected human colorectal tumors and animal models. Mechanistically, the incorporation of γ-glutamyl group facilitates the rapid internalization of Poly-g-BAT, and these internalized nanoprobes can be subsequently activated by intracellular NAD(P)H: quinone oxidoreductase-1 to release near-infrared fluorophores. As a result, Poly-g-BAT can achieve a superior tumor-to-normal ratio (TNR) up to 12.3 and enable a fast visualization (3min after in situ spraying) of tumor boundaries in the xenograft tumor models, Apcmin/+ mice models and fresh human tumor tissues. In addition, Poly-g-BAT is capable of identifying minimal premalignant lesions via intravenous injection.

Direct Implantation of Patient Brain Tumor Cells into Matching Locations in Mouse Brains for Patient-Derived Orthotopic Xenograft Model Development.

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

Abstract 237: Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and there is a critical need for more effective treatments. The development of new therapies, especially those aiming to modulate the tumor microenvironment (TME), is limited by the lack of in vitro and in vivo models that recapitulate the complexity of the TME and spatial architecture of HCC. Here, we aimed to establish an HCC model to assess the treatment response and hepatotoxicity of selected drugs by ex vivo culturing fresh human liver tumor tissue and adjacent normal tissue, respectively. Materials and Methods: Fresh human liver tissues, including 5 malignant and 20 adjacent-normal, were obtained from surgical resections. PDX tumors were generated from patient-derived organoids. Tissue resections were cut into ultra-thin slices (from 150µm to 350µm) using a vibratome. Related culture conditions such as high oxygen partial pressure, air-liquid interface (ALI) culture, artificial extracellular matrix (AEM), and delay duration were evaluated to optimize the protocol. As a readout, cell viability was quantified by an expert pathologist using Hematoxylin/Eosin staining. The preservation and complexity of the TME were evaluated by immunohistochemistry or flow cytometry using specific antibodies for each cell population (T cells, anti-CD3; stroma cells, a-SMA; endothelial cells, CD34; Kupffer cells, CD68). Liver cell function was tested by measuring ALT/AST activity, albumin release, and CYP340 activity. Results: The highest tissue viability was observed in normal and tumor tissue with a thickness of 150µm and 250µm, respectively. Compared to standard conditions, the combination of ALI and high-oxygen partial pressure conditions dramatically improved tissue viability, with most of the samples having 70% and 50% cell viability at days 3 and 5 of ex vivo culture, respectively (p<0.05). AEM could prevent the efflux of cellular components (p=0.016). The impact of the time delay between the liver resection and the start of slicing was minimal when the slicing occurred within two hours after hepatectomy (p<0.05). Key cellular components including hepatocytes, T cells, fibroblasts, and Kupffer cells also remained better preserved throughout the 5-day culture period in optimized conditions. Conclusion: Our study has successfully optimized the ex vivo tissue culture model of liver tissue, capable of maintaining the tissue for several days while preserving the unique TME. These results highlight the potential of using the ex vivo tissue culture model in the preclinical setting to further investigate and evaluate TME-related treatments and their hepatotoxicity in HCC tissues. Citation Format: Huiche Feng, Miriam Cieri, Cinzia Esposito, Karolina Guja-Jarosz, Daniela Liberati, Lukas Bubendorf, Savas Deniz Soysal, Mairene Coto-Llerena, Luigi Maria Terracciano, Otto Kollmar, Salvatore Piscuoglio. Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 237.

Abstract 3187: Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy

Abstract Background: Activating the innate immune pathway of stimulator of interferon genes (STING) can elicit potent anti-tumor immunity via the production of type-I interferons (IFN-I). However, cyclic-dinucleotide (CDN) STING agonists have undesirable pharmacological properties. Most CDNs in clinical development are administered intratumorally, thus precluding their utility against advanced cancer. Here, we report the development of a new nanoparticle system for the systemic delivery of CDN STING agonists with potent efficacy, favorable pharmaceutical properties, and acceptable safety profiles. Methods: Various metal ions were screened for synergy with CDNs for IFN-I response from bone marrow-derived dendritic cells. Lipid nanoparticles carrying CDN and manganese ions (termed SNP) were synthesized. Mice bearing syngenetic tumors (including CT26 and B16F10) as well as genetically engineered mouse models were treated by intravenous administration of SNP, followed by tumor monitoring and immune profiling of the tumor microenvironment. White New Zealand rabbits bearing VX2 tumors as well as healthy mongrel dogs were treated with SNP and evaluated. Lastly, human tumor biopsies derived from head and neck squamous cell carcinoma (HNSCC) patients were incubated with SNP or soluble STING agonists and analyzed for immune activation. Results: We identified that Mn2+ achieved strong synergy with CDN STING agonists for inducing IFN-I production. SNP carrying CDN and Mn2+ was dosed intravenously in mice, leading to robust IFN-I response, remodeling of the immunosuppressive tumor microenvironment, and expansion of anti-tumor CD8+ T cells. Intravenous SNP therapy resulted in robust anti-tumor efficacy in multiple murine tumor models, including CT26, B16F10, and a genetically engineered mouse model of MMTV-PyMT triple-negative breast cancer. We have also observed robust anti-tumor efficacy of SNP against VX2 squamous carcinomas in rabbits and have demonstrated the safety of intravenous SNP therapy in healthy dogs. Mechanistically, the efficacy of SNP therapy was dependent on the host STING expression but independent of the tumor STING expression. In addition, the anti-tumor efficacy of SNP was decreased in mice lacking Ifnar1 or Ifngr1 expression in the B16F10 melanoma model, showing the crucial effects of IFN-I and IFN-II responses. Moreover, SNP treatment led to robust IFN-I responses from fresh human tumor biopsies derived from HNSCC patients. Conclusions: SNP empowers highly effective systemic cancer immunotherapy via nanotechnology. It also underscores the potential of utilizing metal ions for immune-mediated disease treatment. Citation Format: Xingwu Zhou, Xiaoqi Sun, Wang Gong, Ziye Wan, Teresa Krieger-Burke, Vilma Yuzbasiyan-Gurkan, Yu Leo Lei, James Moon. Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3187.

Reversing immunosuppression in the tumor microenvironment of fibrolamellar carcinoma via PD-1 and IL-10 blockade

Fibrolamellar carcinoma (FLC) is a rare liver tumor driven by the DNAJ-PKAc fusion protein that affects healthy young patients. Little is known about the immune response to FLC, limiting rational design of immunotherapy. Multiplex immunohistochemistry and gene expression profiling were performed to characterize the FLC tumor immune microenvironment and adjacent non-tumor liver (NTL). Flow cytometry and T cell receptor (TCR) sequencing were performed to determine the phenotype of tumor-infiltrating immune cells and the extent of T cell clonal expansion. Fresh human FLC tumor slice cultures (TSCs) were treated with antibodies blocking programmed cell death protein-1 (PD-1) and interleukin-10 (IL-10), with results measured by cleaved caspase-3 immunohistochemistry. Immune cells were concentrated in fibrous stromal bands, rather than in the carcinoma cell compartment. In FLC, T cells demonstrated decreased activation and regulatory T cells in FLC had more frequent expression of PD-1 and CTLA-4 than in NTL. Furthermore, T cells had relatively low levels of clonal expansion despite high TCR conservation across individuals. Combination PD-1 and IL-10 blockade signficantly increased cell death in human FLC TSCs. Immunosuppresion in the FLC tumor microenvironment is characterized by T cell exclusion and exhaustion, which may be reversible with combination immunotherapy.

Abstract LB_B16: Quantification of immune-oncology drug responses in autologous co-cultures of ex vivo patient tumor tissues with PBMCs

Abstract Introduction Immuno-oncology drug treatments, including immune checkpoint inhibitors, represent a huge promise for cancer patients and have been clinically approved for some cancer indications. However, patient response rates stay relatively low (about 10-20% responders across indications) hampering the success of such drugs in clinical trials. In addition, current in vitro and in vivo pre-clinical models often lack the (immunological) complexity necessary to predict the clinical effectiveness of such drugs or drug combinations. To bridge this gap in translational models, we have recently developed the 3D Ex vivo Patient Tissue (EVPT) platform. The EVPT platform is leveraging short-term 3D ex vivo tumor culture system containing micro-tissues generated from fresh human tumor material and high content imaging analysis. The platform is preserving the innate tumor microenvironment including resident immune cells, which are crucial for addressing immunological drug responses. In this study we aimed to further boost immune compartment of the assay by addition of autologous immune cells (PBMCs) isolated from whole blood of the patient in parallel with ex vivo tumor processing. Methods Patient tumor tissues from resected NSCLC and muscle-invasive bladder cancer (MIBC) tumors and matching peripheral blood were obtained from commercial tissue providers and processed within 24 hours to preserve the TME. PBMCs were isolated from whole blood and pre-stained with cell tracker. Fresh, minimally processed tumor material was embedded in a protein-rich hydrogel with or without matching PBMCs and exposed to mono- and combination immunotherapies at various doses. Next, the phenotypic screening using our proprietary automated HCI analysis platform was performed. Where applicable, IHC biomarker, flow cytometry and cytokine measurements were performed on fixed tissues or culture supernatants. Results 3D ex vivo co-cultures were executed in 22 NSCLC and 5 MIBC patient samples. Immunological treatment responses varied between tested patients. When comparing co-culture to mono-culture conditions, one third of the samples showed baseline killing associated with PBMCs addition. About 20% of tissues from both indications responded to antiPD1 treatment (Pembrolizumab) and on average 50% of tissues responded to immunostimulatory control SEA. Overall best effects were observed for one test article that was used in three doses in combination with antiPD1. Five out five tested NSLCLC samples showed functional responses in these conditions as wells as two out five MIBC samples. In addition, it induced immunostimulatory effects on the PBMCs in the assay – increased numbers and tumor infiltration rates compared to vehicle control. Conclusions 3D Ex vivo Patient Tissue platform combined with autologous PBMCs offers supplementary readouts such as tumor infiltration and immune cell proliferation while providing TME-rich environment for functional immune-oncology drug testing. Citation Format: Niels Meesters, Sander Basten, Ezgi Kaya Aksoy, Mariusz Madej, Kuan Yan, Emma Spanjaard, Leo S. Price, Nataliia Beztsinna. Quantification of immune-oncology drug responses in autologous co-cultures of ex vivo patient tumor tissues with PBMCs [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B16.

Enhancing STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy.

2571 Background: Activating the innate immune pathway of stimulator of interferon genes (STING) can elicit potent anti-tumor immunity via the production of type-I interferons (IFN-I). However, cyclic-dinucleotide (CDN) STING agonists have undesirable pharmacological properties. Most CDNs in clinical development are administered intratumorally, thus precluding their applications in treating metastasis, hematologic malignancies, and hard-to-reach solid tumors. Here, we report the development of a new nanoparticle system for the systemic delivery of CDN STING agonists with potent efficacy, favorable pharmaceutical properties, and acceptable safety profiles. Methods: Nutritional metal ions play crucial roles in modulating immune pathways, which could potentially be used for enhancing STING activation. We screened different metal ions and found that manganese ion (Mn2+) achieved strong synergy with CDN STING agonists for inducing IFN-I production. We further developed a new nanoparticle system for co-delivery of Mn2+ and CDN for systemic cancer immunotherapy. Results: Mn2+ induced up to 77-fold enhancement in CDN-mediated STING activation. Mixing CDNs with Mn2+ resulted in the self-assembly of nanocrystals, and the lipid layer coating led to uniform nanoparticles. The resulting STING-activating CDN-Mn nanoparticle (SNP) given intravenously elicited IFN-I responses, attenuated the suppressive functions of myeloid cells, remodeled the immunosuppressive tumor microenvironment, and expanded CD8+ T cells for sustained tumor control. Intravenous SNP therapy resulted in robust anti-tumor efficacy in multiple murine tumor models, including a genetically engineered mouse model of MMTV-PyMT triple-negative breast cancer. We have also demonstrated the robust anti-tumor efficacy of SNP against VX2 head and neck squamous cell carcinomas (HNSCC) in New Zealand white rabbits. Mechanistically, the efficacy of intravenous SNP therapy was dependent on the host STING expression but independent of the tumor STING expression. In addition, the anti-tumor efficacy of SNP was decreased in mice lacking Ifnar1 or Ifngr1 expression in the B16F10 melanoma model, showing the crucial effects of IFN-I and IFN-II responses. Lastly, SNP was effectively uptaken by human PBMCs and elicited IFN-I responses when injected into fresh human tumor biopsies derived from HNSCC patients. Conclusions: SNP empowers highly effective systemic cancer immunotherapy via nanotechnology. It also underscores the potential of utilizing metal ions for immune-mediated disease treatment.

Recent Advancement in Breast Cancer Research: Insights from Model Organisms-Mouse Models to Zebrafish.

Animal models have been utilized for decades to investigate the causes of human diseases and provide platforms for testing novel therapies. Indeed, breakthrough advances in genetically engineered mouse (GEM) models and xenograft transplantation technologies have dramatically benefited in elucidating the mechanisms underlying the pathogenesis of multiple diseases, including cancer. The currently available GEM models have been employed to assess specific genetic changes that underlay many features of carcinogenesis, including variations in tumor cell proliferation, apoptosis, invasion, metastasis, angiogenesis, and drug resistance. In addition, mice models render it easier to locate tumor biomarkers for the recognition, prognosis, and surveillance of cancer progression and recurrence. Furthermore, the patient-derived xenograft (PDX) model, which involves the direct surgical transfer of fresh human tumor samples to immunodeficient mice, has contributed significantly to advancing the field of drug discovery and therapeutics. Here, we provide a synopsis of mouse and zebrafish models used in cancer research as well as an interdisciplinary 'Team Medicine' approach that has not only accelerated our understanding of varied aspects of carcinogenesis but has also been instrumental in developing novel therapeutic strategies.

Abstract 668: The translational biology of small molecule GPR65 inhibitors: shared effects between mouse models and human primary tumors highlight the unique transformative potential of targeting a genetically validated innate immune checkpoint

Abstract Whilst the advent of Immune Checkpoint Blockade has revolutionized the management of cancer, a significant proportion of patients have limited or absent response to these therapies. A key cause of this immune insensitivity is the hostile solid tumor microenvironment (TME) dominated by immunosuppressive myeloid cells. We previously identified the acid sensing G protein coupled receptor (GPCR), GPR65, as a primary determinant of these suppressive cells. In mice, genetic deletion of Gpr65 or oral administration of small molecule GPR65 inhibitors in vivo causes a profound repolarization of immunosuppressive tumor associated macrophages, an increase in infiltrating effector cells and potent anti tumor effects in syngeneic models. In TCGA data, across all tumors, patients homozygous for a hypomorphic coding variant in GPR65 (I231L) show increased overall survival, providing compelling genetic evidence of the clinical potential of GPR65 inhibition. To further explore the translational potential of GPR65 we employed a range of techniques to define the human biology of this receptor in different contexts. At the mechanistic level, single cell RNA sequencing (scRNAseq) of human PBMCs obtained from healthy donors demonstrated a pronounced effect of low pH on the myeloid compartment, with a clear polarization of these cells toward an immunosuppressive character and modulation of GPR65 expression. In parallel, pharmacological inhibition of GPR65 in human monocyte derived macrophages exposed to low pH demonstrated that equivalent gene expression changes are primarily due to GPR65 activation. To examine the relevance of these findings to the intact acidic human TME, we performed studies in fresh primary human tumor histocultures from clear cell renal cell carcinoma (ccRCC) patients with immunohistochemically confirmed high macrophage infiltration and carbonic anhydrase 9 (CA9) expression. In these cultures, GPR65 inhibition caused a dose dependent suppression of a geneset closely overlapping with that modulated by GPR65 in primary macrophages. Furthermore, we observed a marked decrease in immune suppressive IL10 secretion with coincident elevation of specific proinflammatory chemokines. Consistent with these findings, in vivo administration of a small molecule GPR65 inhibitor elicited similar changes in human CA9 expressing RCC PDX tumors implanted in myeloid boosted CD34+ stem cell engrafted NCG mice. In summary, inhibition of GPR65 provides a unique and genetically validated approach to favorably modify the immunosupressive TME with features highly conserved between mouse and human contexts. We propose that GPR65 inhibition holds significant clinical promise, with specific evidence around ccRCC as a potential standout indication. Citation Format: Barbara Cipriani, Alastair Corbin, David Miller, Alan Naylor, Faraz Khan, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Mussa Quareshy, Anastasia Nika, Preeti Singh, Gavin Knox, Darryl Turner, Satish Sankaran, Nandini Pal Basak, Toszka Bohn, Tobia Bopp, Surya Koturan, Bo Sun, Benjamin Fairfax, Tom McCarthy, Stuart Hughes. The translational biology of small molecule GPR65 inhibitors: shared effects between mouse models and human primary tumors highlight the unique transformative potential of targeting a genetically validated innate immune checkpoint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 668.

Translational albumin nanocarrier caging photosensitizer for efficient cancer photodynamic therapy.

It still remains a great challenge to efficiently treat malignant cancers which severely threaten human health. Photodynamic therapy (PDT) as a localized therapeutic modality has improved the therapeutic efficacy via chemical damage through reactive oxygen species (ROS). However, their efficacy is severely hampered by insufficient targeted delivery of photosensitizers owing to the lack of suitable carrier with facile preparation process and the clinical applicability. Herein, we applied clinically approved human serum albumin as the nanoreactor to encapsulate photosensitizers Chlorin e6 (Ce6) for enhancing their tumor accumulation and subsequently potent PDT effect against bladder cancer models. Albumin-loaded Chlorin e6 nanoparticles (CA-NPs) with rational nanoscale size exhibit increased reactive oxygen species production and excellent resistance to photobleaching. Moreover, CA-NPs could be efficiently internalized by tumor cells and locate in the lysosome, while they rapidly translocate to cytosol after irradiation to induce remarkable cytotoxicity (IC50 ∼5.8μg/ml). Furthermore, CA-NPs accumulate effectively in tumor tissue to afford total eradication of murine bladder tumor after single injection. More importantly, we also evidence the superior PDT effect in fresh human bladder tumor tissues via abundant reactive oxygen species generation and subsequent cell apoptosis. These findings demonstrate that human serum albumin acts as a universal tool to load small organic photoactivatable molecule with remarkable effectiveness and readiness for clinical translation.

A Therapeutically Actionable Protumoral Axis of Cytokines Involving IL-8, TNFα, and IL-1β.

IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007.

Abstract 3826: A therapeutically actionable pro-tumoral axis of cytokines involving interleukin-8, TNFα and IL-1β

Abstract Background: Interleukin-8 produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. An important question is what are the mechanisms that control IL-8 transcription in tumors. Various factors have been described but the main common denominator is NF-кB activation. In this regard, considering inflammatory mediators present in the tumor and whose receptors act as NF-кB inducers, TNFα and IL-1β became obvious candidates. In this study, we provide multiple lines of evidence that TNFα and IL-1β are able to elicit functional IL-8 from malignant cells in a manner that can be inhibited by clinically grade blockade agents. Methods: Short-term cultured human tumor cell lines, fresh human tumor explants and primary organoids were stimulated with TNFα or IL-1β to test for IL-8 induction. Moreover, syngeneic and xenografted tumors were also used to study IL-8 induction in response to TNFα or to IL-1β. Correlation between these cytokines and IL-8 transcripts were studied in TCGA and other databases to search. Results: We have been able to correlate IL-8 mRNA levels with IL-1β and TNFα transcripts in human cancers. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids and fresh human tumor explants. Although IL-8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in several mouse tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα-blockers infliximab and etanercept, or the IL-1β inhibitor anakinra were able to interfere with this pathogenic cytokine loop. Moreover, treatment with checkpoint inhibitors augments TNFα in syngeneic and xenografted tumors thereby turning on this immunosuppressive chemokine-driven axis. However, we have demonstrated that co-blockade of TNFα and IL-1β could equally enhance anti-tumor efficacy of αPD1+αCTLA-4 regimens as CXCR1/CXCR2 inhibitors, reparixin and AZD5069, did. Conclusions: In this study, we have identified TNFα and IL-1β as major inducers of IL-8 transcription on human cancers. In keeping with these findings, both TNFα and IL-1β induce functional IL-8 release from all tested human tumor cell lines representing a variety of tissue origins, as well as from primary organoids and fresh human cancer explants. In all these cases, including xenografted human tumors, IL-8 induction could be blunted by the pharmacological blockade of TNFα or IL-1β. Finally, inhibition of this pathogenic cytokine loop could increase the anti-tumoral effect of checkpoint inhibitors. Citation Format: Irene Olivera, Rebeca Sanz-Pamplona, Elixabet Bolaños, Inmaculada Rodriguez, Iñaki Etxeberria, Assunta Cirella, Saray Garasa, Itziar Migueliz, Iñaki Eguren, Miguel F. Sanmamed, Javier Glez-Vaz, Arantza Azpilikueta, Maite Alvarez, Maria C. Ochoa, Carlos E. De Andrea, Pedro Berraondo, Álvaro Teijeira, Ignacio Melero. A therapeutically actionable pro-tumoral axis of cytokines involving interleukin-8, TNFα and IL-1β [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3826.

Abstract 5613: Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures, and inhibits tumor growth in in vivo murine tumor models

Abstract VSIG4 is a B7 family related protein with known roles as a complement receptor involved in pathogen clearance as well as a negative regulator of T cell activation by an undetermined mechanism. VSIG4 is expressed in tumor associated macrophages (TAMs) with exquisite specificity. In cancer, increased expression of VSIG4 has been associated with worse survival in multiple indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, and glioma, suggesting an important role in tumor immune evasion. Based upon computational analysis of transcript data across thousands of primary cancer and normal tissue samples, we hypothesized that VSIG4 has an important regulatory role in promoting M2-like immune suppressive macrophages in the tumor microenvironment, and that targeting VSIG4 via a monoclonal antibody could relieve VSIG4-mediated macrophage suppression by repolarizing TAMs to an inflammatory phenotype capable of coordinating an anti-tumor immune response. The ability of anti-VSIG4 antibodies to repolarize M2-like macrophages and induce broad immune activation was studied in in vitro, ex vivo, and in vivo models. In in vitro primary cell assays, anti-VSIG4 upregulated pro-inflammatory cytokines and chemokines in M-CSF plus IL-10 driven monocyte-derived M2c macrophages, inducing a shift toward an M1-like phenotype. In a multicellular assay system, blockade of VSIG4 on M2c macrophages co-cultured with autologous T cells in the presence of SEB activation upregulated the expression of both pro-inflammatory myeloid-derived cytokines and T cell-derived cytokines, suggesting that macrophage repolarization in vitro can induce T cell activation. To assess these observations in a relevant translational model, fresh, patient-derived tumor samples were treated ex vivo with anti-VSIG4. Across multiple tumor types, anti-VSIG4 treatment resulted in a significant upregulation of cytokines involved in TAM repolarization and T cell activation, and chemokines involved in immune cell recruitment, at levels generally greater than observed by treatment with anti-PD-1. To determine whether targeting VSIG4 can lead to an anti-tumor effect in vivo, syngeneic mouse models were dosed with anti-mouse VSIG4 antibodies. Tumor growth inhibition was observed in syngeneic mouse models dosed with anti-VSIG4 alone and in combination with anti-PD-1. Tumor growth inhibition was partially reversed in studies where CD8+ T cells were depleted, demonstrating that targeting VSIG4 on macrophages can elicit a coordinated attack on tumors in vivo through both innate and adaptive immune responses. Taken together, these data suggest that VSIG4 represents a promising new target capable of stimulating an anti-cancer response via multiple key immune mechanisms. Citation Format: Stephen Sazinsky, Ani Nguyen, Mohammad Zafari, Boris Klebanov, Jessica Ritter, Veronica Komoroski, Denise Manfra, Igor Feldman, Tatiana Novobrantseva. Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures, and inhibits tumor growth in in vivo murine tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5613.

MP06-13 TARGETING THE ANDROGEN RECEPTOR AXIS TO IMPROVE RESPONSE TO BLADDER CANCER IMMUNOTHERAPY

You have accessJournal of UrologyCME1 May 2022MP06-13 TARGETING THE ANDROGEN RECEPTOR AXIS TO IMPROVE RESPONSE TO BLADDER CANCER IMMUNOTHERAPY Typhaine Gris, Marjorie Besançon, France-Hélène Joncas, Valérie Picard, Alain Bergeron, Yves Fradet, and Paul Toren Typhaine GrisTyphaine Gris More articles by this author , Marjorie BesançonMarjorie Besançon More articles by this author , France-Hélène JoncasFrance-Hélène Joncas More articles by this author , Valérie PicardValérie Picard More articles by this author , Alain BergeronAlain Bergeron More articles by this author , Yves FradetYves Fradet More articles by this author , and Paul TorenPaul Toren More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Men are 3-4 times more likely to be diagnosed with bladder cancer (BCa) than women, who often have more aggressive tumors. Intravesical instillation of BCG is one of the first immunotherapies in widespread use and is given to patients with non-muscle invasive bladder cancer (NMIBC). Newer PD-1/PD-L1 checkpoint inhibitors now also have proven efficacy against BCa. Nonetheless, approximately 35% of patients fail to respond to BCG, with response rates to checkpoint immunotherapy much lower. Based on prior work suggesting the importance of the androgen receptor (AR) in BCa, we investigated AR-targeting as a novel strategy to improve the response to BCa immunotherapy in the MBT-2 murine BCa model. METHODS: Subcutaneous injection of 106 MBT-2 cells into male and female C3H mice was performed, with tumors harvested at tumor size limits. The AR antagonist enzalutamide (ENZ) was given in alone or combination with anti-PD-1 or intra-tumoral BCG treatments as previously described. Selected human NMIBC were collected following transurethral resection. After digestion of fresh human and murine tumors, flow cytometry analyses characterized the immune cells present. The anti-proliferative effect of AR antagonists against human and murine BCa cell lines was tested in vitro. RESULTS: The CD45+/CD45- ratio, extent of tumor infiltrating lymphocytes, and expression of immune checkpoint markers from the MBT-2 model is comparable to the immune composition of NMIBC tumors resected from patients. Moreover, the model also recapitulates previously observed human tumor immune cell gender differences. AR antagonist ENZ demonstrated anti-proliferative effects in lower grade human and murine BCa cell lines, while limited differences were seen in human high grade BCa cell lines. While ENZ alone had no effect in vivo, its use in combination with either anti-PD-1 or BCG treatment in male mice synergized to improve response rates. Complete response was confirmed by re-challenge of mice, demonstrating immune memory. Notably, the proportion of complete responses in male mice treated with the combination treatment is similar to that observed in female mice with either immunotherapy alone. Flow cytometry analyses showed that these therapeutic benefits in male mice are related to an increase in pro-inflammatory tumor infiltrating dendritic cells as well as a decrease in anti-inflammatory tumor infiltrating dendritic cells and myeloid-derived suppressor cells. CONCLUSIONS: Our results suggest that combining AR antagonism with immunotherapy in male BCa patients may potentiate the antitumor immune response and increase response rates. Accordingly, a Phase II trial to evaluate the efficacy of AR antagonism in combination with BCG to decrease NMIBC tumor recurrences in men is planned. Further, the MBT-2 murine model appears relevant to investigate immunologic BCa sex differences. Source of Funding: Cancer Research Society (Upcycle Grant) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e82 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Typhaine Gris More articles by this author Marjorie Besançon More articles by this author France-Hélène Joncas More articles by this author Valérie Picard More articles by this author Alain Bergeron More articles by this author Yves Fradet More articles by this author Paul Toren More articles by this author Expand All Advertisement PDF DownloadLoading ...

Abstract P105: Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures

Abstract VSIG4 (V-set immunoglobulin-domain-containing 4) is a B7 family related protein with known roles as a complement receptor involved in pathogen clearance, via interactions with C3 fragments, as well as a negative regulator of T cell activation by an undetermined mechanism. VSIG4 is expressed in tumor associated macrophages with exquisite specificity. In cancer, increased expression of VSIG4 has been associated with worse survival in multiple indications, including non-small cell lung cancer, multiple myeloma, ovarian cancer, stomach cancer and glioma, suggesting an important role in tumor immune evasion. Based upon computational analysis of transcript data across thousands of primary cancer and normal tissue samples, we hypothesized that VSIG4 has an important regulatory role in promoting M2-like immune suppressive macrophages in the tumor microenvironment, and that targeting VSIG4 via a monoclonal antibody could relieve VSIG4-mediated macrophage suppression by repolarizing tumor associated macrophages (TAMs) to an inflammatory phenotype capable of coordinating an anti-tumor immune response. Through a series of in vitro and ex vivo assays we demonstrate that anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Anti-VSIG4 antibodies upregulate pro-inflammatory cytokines in M-CSF plus IL-10 driven monocyte-derived M2c macrophages, as well as in TAM-like macrophages in vitro derived from monocytes cultured in the presence of ascites fluid from ovarian cancer patients. To determine whether anti-VSIG4-induced macrophage repolarization can activate T cells, monocyte-derived M2c macrophages were co-cultured with autologous T cells in the presence of staphylococcal enterotoxin B (SEB) activation and anti-VSIG4 antibodies. Here, anti-VSIG4 antibodies upregulate both pro-inflammatory myeloid-derived cytokines (GM-CSF) and T cell-derived cytokines (IFNγ). To extend these observations to a relevant translational model, we treated fresh, patient-derived tumor samples with anti-VSIG4 antibodies and relevant controls ex vivo. Across numerous patient-derived samples, which included multiple tumor types, anti-VSIG4 treatment resulted in a significant upregulation of cytokines involved in TAM repolarization and T cell activation, and chemokines involved in immune cell recruitment, at levels greater than observed by treatment with anti-PD-1 or a clinical macrophage repolarizing agent (anti-ILT-4). Taken together, these data suggest that VSIG4 represents a promising new target capable of stimulating an anti-cancer response via multiple key immune mechanisms. Citation Format: Steve Sazinsky, Ani Nguyen, Mohammad Zafari, Ryan Phennicie, Joe Wahle, Veronica Komoroski, Kathryn Rooney, Craig Mizzoni, Boris Klebanov, Jessica Ritter, Denise Manfra, Igor Feldman, Tatiana Novobrantseva. Targeting VSIG4, a novel macrophage checkpoint, repolarizes suppressive macrophages which induces an inflammatory response in primary cell in vitro assays and fresh human tumor cultures [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P105.

Molecular and genomic characterisation of a panel of human anal cancer cell lines

Anal cancer is a rare disease that has doubled in incidence over the last four decades. Current treatment and survival of patients with this disease has not changed substantially over this period of time, due, in part, to a paucity of preclinical models to assess new therapeutic options. To address this hiatus, we set-out to establish, validate and characterise a panel of human anal squamous cell carcinoma (ASCC) cell lines by employing an explant technique using fresh human ASCC tumour tissue. The panel of five human ASCC cell lines were validated to confirm their origin, squamous features and tumourigenicity, followed by molecular and genomic (whole-exome sequencing) characterisation. This panel recapitulates the genetic and molecular characteristics previously described in ASCC including phosphoinositide-3-kinase (PI3K) mutations in three of the human papillomavirus (HPV) positive lines and TP53 mutations in the HPV negative line. The cell lines demonstrate the ability to form tumouroids and retain their tumourigenic potential upon xenotransplantation, with varied inducible expression of major histocompatibility complex class I (MHC class I) and Programmed cell death ligand 1 (PD-L1). We observed differential responses to standard chemotherapy, radiotherapy and a PI3K specific molecular targeted agent in vitro, which correlated with the clinical response of the patient tumours from which they were derived. We anticipate this novel panel of human ASCC cell lines will form a valuable resource for future studies into the biology and therapeutics of this rare disease.

Firefighters for the Wrong Type of Inflammation in Tumors

In this issue of Cancer Discovery, Pelly and colleagues show that inhibition of prostaglandin E2 synthesis or its activity on EP2 and EP4 receptors synergizes with anti-PD-1 immunotherapy and triggers a potent intratumoral IFNγ response in mouse models and in fresh surgical human tumor explants. This therapeutic strategy is in line with other interventions that aim at fostering immunotherapy by means of quenching protumor inflammation.See related article by Pelly et al., p. 2602.

Blocking siglec-10hi tumor-associated macrophages improves anti-tumor immunity and enhances immunotherapy for hepatocellular carcinoma

BackgroundTumor-associated macrophages (TAMs) promote key processes in the modulation of tumor microenvironment (TME). However, the clinical significance of heterogeneous subpopulations of TAMs in hepatocellular carcinoma (HCC) remains unknown.MethodsHCC tissues from Zhongshan Hospital and data from The Cancer Genome Atlas were obtained and analyzed. Immunohistochemistry and flow cytometry were performed to detect the characteristics of sialic acid-binding immunoglobulin-like lectin 10high (Siglec-10hi) TAMs and explore their impact on the TME of HCC. The effect of Siglec-10 blockade was evaluated in vitro based on fresh human tumor tissues.ResultsOur data revealed that Siglec-10 was abundant in a large proportion of HCC specimens and prominently distributed on macrophages. Kaplan–Meier curves and Cox regression analysis showed that intratumoral Siglec-10+ cell enrichment was associated with unfavorable prognosis in patients with HCC. Notably, multiple anti-inflammatory cytokines and inhibitory receptors were enriched in Siglec-10hi TAMs. RNA sequencing data also revealed that numerous M2-like signaling pathways were significantly upregulated in Siglec-10hi TAMs. High infiltration of Siglec-10hi TAMs was associated with impaired CD8+ T cell function in HCC. Of note, blocking Siglec-10 with the competitive binding antibody Siglec-10 Fc led to decreased expression of immunosuppressive molecules and increased the cytotoxic effects of CD8+ T cells against HCC cells. Moreover, blocking Siglec-10 promoted the anti-tumor efficacy of the programmed cell death protein 1 (PD-1) inhibitor pembrolizumab.ConclusionsSiglec-10hi TAMs are associated with immune suppression in the TME, and indicate poor prognosis in patients with HCC. Targeting Siglec-10hi TAMs may serve as a promising immunotherapy approach for HCC.

A biheteroaryl-bridged fluorescence probe enables lipid droplets-specific bioimaging and photodynamic therapy in clinical clear cell renal cell carcinoma

Lipid droplets (LDs) targeted fluorescence probe have been believed to be an effective tool for diagnosis and therapy of some tumors with abnormal lipid metabolism, while there are very few probes with simultaneous high efficiency in LDs imaging and inhibiting tumor proliferation. Herein we present a biheteroaryl-bridged fluorescence probe with aggregation-induced emission (AIE) characteristic, which show dual functions of LDs-specific bioimaging and photodynamic therapy (PDT) based on human clear cell renal cell carcinoma (ccRCC) tumor cells and clinical samples, both rich in LDs. The highly LDs-specific targeting capability, good biocompatibility, excellent photostability and imaging resolution of the AIE probe in ccRCC tumor cells make it considerably attractive to commercially available LDs-specific dyes. Notably, the fluorescence imaging in fresh human ccRCC tumor tissues further proves its outstanding LDs-targeting specificity and demonstrates its power as a bio-probe for clinical diagnosis in vitro and in vivo. Confirmatory experiments suggest a superior PDT efficacy of the AIE probe in ccRCC tumor cells in comparison to those LDs-poor tumor cells and normal cells without LDs. Mechanism studies further reveal the presented AIE probe enables to induce the DNA damage and cell apoptosis by reactive oxygen species generation, resulting in desirable therapeutic efficiency for clinical ccRCC tumor cells upon visible light irradiation.