Successful Therapy Research Articles

Abstract 6530: TREM1 inhibition: A novel approach to suppress tumor growth and enhance immune checkpoint therapy

Abstract In the context of cancer, inflammation is a multifaceted contributor to tumorigenesis. The Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) is recognized for its role in amplifying inflammatory responses, yet its specific pro-tumorigenic mechanisms have remained elusive. This study employs Trem1-deficient mice and a novel inhibitor, VJDT, to explore the therapeutic potential of targeting TREM1 in cancer. Both genetic silencing and pharmacological inhibiton of Trem1 led to remarkable reductions in tumor growth in mouse models of melanoma (B16F10) and fibrosarcoma (MCA205). In-depth analysis using single-cell RNA sequencing unveiled that TREM1 deficiency resulted in a decrease in the frequency and suppressive capabilities of myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment, concomitant with an increase in cytotoxic CD8+ T cells and enhanced T cell activation. Furthermore, TREM1 inhibition not only curtailed tumor growth but also potentiated the effectiveness of anti-PD-1 therapy, primarily by mitigating MDSC frequency and reversing T cell exhaustion. Notably, in melanoma xenografts from patient samples, pharmacological TREM1 inhibition via VJDT treatment significantly downregulated critical oncogenic pathways linked to cell proliferation, migration, and survival. This research underscores the pivotal role of TREM1, expressed both within cancer cells and the tumor microenvironment, in driving cancer progression. Consequently, targeting TREM1 for therapeutic intervention holds promise for reshaping the immunosuppressive tumor milieu and augmenting the success of immune checkpoint therapy. Citation Format: Ashwin Ajith, Kenza Moamouni, Daniel D Horuzsko, Anatolij Horuzsko. TREM1 inhibition: A novel approach to suppress tumor growth and enhance immune checkpoint therapy [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 6530.

Methamphetamine Enhancement of HIV-1 gp120-Mediated NLRP3 Inflammasome Activation and Resultant Proinflammatory Responses in Rat Microglial Cultures.

Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders (HANDs) remain prevalent in HIV-1-infected individuals despite the evident success of combined antiretroviral therapy (cART). The mechanisms underlying HAND prevalence in the cART era remain perplexing. Ample evidence indicates that HIV-1 envelope glycoprotein protein 120 (gp120), a potent neurotoxin, plays a pivotal role in HAND pathogenesis. Methamphetamine (Meth) abuse exacerbates HANDs, but how this occurs is not fully understood. We hypothesize that Meth exacerbates HANDs by enhancing gp120-mediated neuroinflammation. To test this hypothesis, we studied the effect of Meth on gp120-induced microglial activation and the resultant production of proinflammatory cytokines in primary rat microglial cultures. Our results show that Meth enhanced gp120-induced microglial activation, as revealed by immunostaining and Iba-1 expression, and potentiated gp120-mediated NLRP3 expression and IL-1β processing and release, as assayed by immunoblotting and ELISA. Meth also augmented the co-localization of NLRP3 and caspase-1, increased the numbers of NLRP3 puncta and ROS production, increased the levels of iNOS expression and NO production, and increased the levels of cleaved gasderminD (GSDMD-N; an executor of pyroptosis) in gp120-primed microglia. The Meth-associated effects were attenuated or blocked by MCC950, an NLRP3 inhibitor, or Mito-TEMPO, a mitochondrial superoxide scavenger. These results suggest that Meth enhances gp120-associated microglial NLRP3 activation and the resultant proinflammatory responses via mitochondria-dependent signaling.

Abstract 6694: Gut microbial drivers of response to bladder-sparing therapy in human muscle-invasive bladder cancer enhance immunotherapy efficacy and limit immunosuppression following fecal microbiome transplantation in vivo

Abstract BACKGROUND: Radiation therapy (RT) is a bladder-sparing option for Muscle-Invasive Bladder Cancer (MIBC), yet encounters a 30% non-response rate, with half of the patients succumbing to metastasis. Preclinical models demonstrate enhanced antitumor responses with RT combined with PD-1/PD-L1 blockade but give limited insight into the determinants of success for such combination therapies (CT). The gut microbiome, pivotal in conditioning local and peripheral immunity, influences PD-1-based immunotherapy efficacy in various cancers. Immune profiling indicates heightened systemic and antitumor immunity in responders with a favorable gut microbiome, enriched in Akkermansia muciniphila, Bifidobacterium, and Faecalibacterium. Oral gavage of immunogenic bacteria further enhances combined RT and anti-PD-L1 therapy. We thus aim to 1) elucidate the role of patients’ microbiome in shaping anti-tumor immune responses to CT, and 2) use its composition as a predictive factor for CT success. METHODS: We performed shotgun metagenomics on microbial DNA from fecal materials of MIBC RT responders (R, n=26) and non-responders (NR, n=11). Fecal homogenates from donors were gavaged into germ-free (GF) mice. Three weeks post-gavage, MB49 tumor cells were subcutaneously delivered to mice, randomized into four groups: control, anti-PD-L1, RT, and RT + anti-PD-L1. Tumors were harvested seven days post-treatment for single-cell RNA-seq and TCR sequencing (10X Genomics), while stool samples were collected weekly for 16S sequencing. RESULTS: Significant gut microbial disparities were observed between R and NR MIBC patients, with particular enrichment in known promoters of anti-PD-L1 response, such as Phocaeicola dorei in R. Transplantation of R microbiome into GF mice resulted in significant increases in predicted survival, tumor growth delay, and counts of effector CD4+ T cell infiltration in the RT+PD-L1 arm. Conversely, NR microbiome transplantation correlated with increased neutrophil and Treg infiltration, abrogating the survival tumor growth delay benefits of the combination arm. Spatial tissue analyses revealed higher neutrophil densities around CD8+ T cells in all NR arms. Single-cell RNA-seq of tumor-infiltrating immune cells unveiled skewed CD4 and CD8 polarization between R and NR in vivo batches. SIGNIFICANCE: To our knowledge, this is the first study to use FMT as a modulator of response in the context of radiation therapy combinations in MIBC. Our results highlight that the gut microbiome alone may condition a favorable immune terrain for combinatorial therapies involving radiation. This offers strong predictive value for personalized therapeutic approaches in selecting patients who will benefit the most from bladder-sparing therapies. Citation Format: Eva Michaud, Sabina Fehric, Cynthia Faubert, Bertrand Routy, Irah King, José Joao Mansure, Wassim Kassouf. Gut microbial drivers of response to bladder-sparing therapy in human muscle-invasive bladder cancer enhance immunotherapy efficacy and limit immunosuppression following fecal microbiome transplantation in vivo [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 6694.

Abstract 750: Specific oral bacterial profile associated with oral HPV status in Puerto Rican People with HIV

Abstract Human papillomavirus (HPV) accounts for more than 70% of oropharyngeal cancers and people with HIV (PWH) are disproportionately affected by HPV infection even with successful antiretroviral therapy. Puerto Rico (PR) is in the top ten in terms of HIV prevalence in the US, and also has disparities in HPV-related malignancies. The oral microbiome can increase the risk of HPV infection and persistence; however, it has not been investigated in the context of HIV in PR. Therefore, we evaluated diversity and composition of the oral bacteriome of 48 virologically suppressed PWH (men and women ≥21 and <64 years old) living in PR in relation to HPV status (HPV+: n=18 vs. HPV-: n=30). We collected saliva (16S rDNA sequencing), oral rinse (HPV genotyping) as well as sociodemographic, lifestyle and behavioral characteristics. Short chain fatty acids (SCFA, acetate, butyrate and propionate) were quantified using gas chromatography and mass spectrometry. Periodontal disease was assessed by a clinical full-mouth periodontal assessment using the CDC/AAP guidelines. All analyses were performed using QIIME2 and R-statistical software. There were no significant differences in alpha diversity (Shannon, observed OTUs, and Faith) between HPV+ and HPV- groups. Absolute CD4 count was not significantly different between HPV+ and HPV- participants (794 cells/µL vs. 735 cells/µL), and did not correlate with either microbial richness or diversity. Higher microbial richness was associated with lower levels of acetate (r=-0.35, p<0.05) and propionate (r=-0.31, p<0.05,). Additionally, higher phylogenetic diversity was also associated with lower levels of acetate (r=-0.38, p<0.05) and propionate (r=-0.35, p<0.05) irrespective of the HPV status. On the other hand, higher levels of butyrate was significantly associated with higher microbial diversity (Shannon index; r=0.33, p< 0.05). Taxonomic analyses showed significantly higher abundance of Dialister and Nanosynbacter genera in HPV+ participants and higher abundance of Rothia genus and Capnocytophaga sputigena in HPV- participants. Overall, we found specific prokaryotic profile and associated metabolites associated with HPV infection, which may suggest that the oral microbiome could influence the natural history of HPV infection via SCFA signaling. Members of the Dialister genus have been found to be prevalent in Hispanic and Black women cervicovaginal microbiota, and as well as in HPV infection and oral cancer. Understanding the underpinning mechanisms of how the oral microbiome can facilitate HPV infection and persistence in PWH is essential to establish targets for early identification and personalized treatment approaches for oral HPV-related malignancies. Citation Format: Yakshi N. Ortiz-Maldonado, Gabriel Borges-Velez, Jurelis Torres-Reyes, Alvin A. Peralta-Betanncourt, Jeannette L. Salgado-Montilla, Jorge Viera-Vera, Maria M. Sanchez-Vazquez, Magaly Martinez-Ferrer, Ana P. Ortiz-Martinez, Ramon F. Gonzalez-Garcia, Josue Perez-Santiago. Specific oral bacterial profile associated with oral HPV status in Puerto Rican People with HIV [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 750.

Biomaterial-mediated intracellular control of macrophages for cell therapy in pro-inflammatory and pro-fibrotic conditions

Macrophages are key modulators of all inflammatory diseases and essential for their resolution, making macrophage cell therapy a promising strategy for regenerative medicine. However, since macrophages change rapidly in response to microenvironmental cues, their phenotype must be controlled post-administration. We present a tunable biomaterial-based strategy to control macrophages intracellularly via small molecule-releasing microparticles. Poly(lactic-co-glycolic acid) microparticles encapsulating the anti-inflammatory and anti-fibrotic drug dexamethasone were administered to macrophages in vitro, with uptake rates controlled by different loading regimes. Microparticle dose and dexamethasone content directly affected macrophage phenotype and phagocytic capacity, independent of particle content per cell, leading to an overall pro-reparative, anti-inflammatory, anti-fibrotic phenotype with increased phagocytic and ECM degrading functionality. Intracellularly controlled macrophages partially maintained this phenotype in vivo in a murine pulmonary fibrosis model, with more prominent effects in a pro-fibrotic environment compared to pro-inflammatory. These results suggest that intracellular control using biomaterials has the potential to control macrophage phenotype post-administration, which is essential for successful macrophage cell therapy.

Abstract 6200: Single-cell analyses unravel novel immune and tumor cell characteristics linked to the response to neoadjuvant immune checkpoint blockade in patients with gastric cancer

Abstract Background: Gastric cancer remains the leading cause of cancer-related deaths worldwide, ranking fifth for incidence and fourth for mortality. Combining neoadjuvant therapy with immune checkpoint inhibitors has demonstrated improved outcomes for patients with gastric cancer. However, responses exhibit heterogeneity, and the understanding of the mediators influencing therapy response is limited. Methods: In this study, we collected 80 blood and tumor samples from 22 gastric cancer patients treated with XELOX/SOX alone or its combination with Nivolumab (anti-PD-1). Paired single-cell RNA-, TCR-, and BCR-sequencing were performed on pre- and post-treatment samples to investigate the dynamics of immune and tumor cells during the development of response/resistance to therapy. Results: Our findings revealed that elevated levels of baseline CD4 and CD8+ tissue-resident memory T (TRM) cells, in activated and stressed states, are associated with a superior response to combined therapy, and treatment further amplifies this cell population in responsive patients. Tracking T cell receptor clones across pre-/post-treated blood and tumor samples disclosed that the expanded clonotypes of TRM cells are exclusively detected in tumors, and considerable preexisting clones contribute to the expansion of these T cell populations following treatment. The simultaneous presence of TRM, B, and dendritic cells in pre-treated tumors predicted a favorable response, and they concertedly expanded in tumors responsive to anti-PD-1 therapy. Conclusions: Our findings highlight the significance of TRM cells in effective responses to anti-PD-1 therapies and indicate that their cellular communication with B and dendritic cells play a pivotal role in shaping the antitumor immunity elicited by combination therapy. Together, this study illuminates vital immune cell subsets during immune checkpoint blockade treatment, provides biological insights into the mechanism of successful combination therapy, and unravels potential targets for improving treatment efficacy in gastric cancer. Citation Format: Enyu Dai, Jiang-Jiang Qin, Can Hu, Zhiyuan Xu, Guangchun Han, Yanshuo Chu, Ruiping Wang, Minghao Dang, Yunhe Liu, Guangsheng Pei, Jiahui Jiang, Fuduan Peng, Yuanyuan Zhang, Andre Luis Faustino, Jaffer A. Ajani, Linghua Wang, Xiangdong Cheng. Single-cell analyses unravel novel immune and tumor cell characteristics linked to the response to neoadjuvant immune checkpoint blockade in patients with gastric cancer [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 6200.

Abstract 2697: Simultaneous activation of innate immune suppressive components impairs antitumor efficacy of immunotherapy against the innate immune system

Abstract Cancer immunotherapy has shown noticeable clinical efficacy in many cancer types, while clinical benefits are only observed in 20-40% of treated patients. Therefore, combination cancer immunotherapy targeting multiple factors that limit antitumor immunity has been tested in the clinic. Accumulating evidence reveals that cancers that harbor limited immune cell infiltration (so-called immunologically cold tumors) respond poorly to current immunotherapy. Preclinical studies using animal models have shown that activation of innate immunity by TLR agonists or STING agonists can successfully remodel the TME to effectively induce antitumor immunity in those immunologically cold tumors. However, unexpectedly, all TLR agonists and STING agonists tested have not shown clinical efficacy in combination with immune checkpoint blockade therapy, including PD-1/PD-L1 blockade therapy. Hence, we addressed the mechanism(s) why stimulators of innate immunity failed to elicit effective antitumor immunity in cancer patients. We then employed OK-432, which reportedly stimulates TLR-2, TLR-4, and/or TLR-9, as OK-432 is widely used in Japan in the clinic. In vitro OK-432 treatment accelerated the maturation DCs, and efficiently induced CD8+ T cells in humans. However, the combination treatment with anti-PD-1 mAb and OK-432 failed to augment antitumor immunity in animal models using anti-PD-1 mAb monotherapy-resistant tumors; the data mirrors the failure of clinical trials of combination therapy with immune checkpoint blockade and TLR agonists or STING agonists in humans. Therefore, using the animal models, we explored the acquired resistance mechanism developed after combination treatment. While OK-432 treatment effectively stimulated DCs, innate immune suppressive cells, such as MDSCs, were concurrently accumulated in the TME. The accumulation of PMN-MDSCs after OK-432 treatment was induced in a CXCL1-CXCR2-dependent fashion. Accordingly, triple combination treatment with a PMN-MDSC inhibitor along with OK-432 and anti-PD-1 mAb attenuated the accumulation of PMN-MDSCs in the TME and improved the antitumor effect of the combination treatment. Taken together, we propose that successful combination treatment stimulating innate immunity against immunogenically cold cancers requires controlling the unwanted co-activation of innate immune suppressive cells. This study clearly demonstrates the mechanism of acquired resistance after administration of reagents that activate innate immunity, such as TLR agonists or STING agonists, and provides the basis for future successful combination therapy. Citation Format: Hitomi Nishinakamura, Sayoko Shinya, Takuma Irie, Shugo Sakihama, Tadaaki Ioroi, Takeo Naito, Keisuke Watanabe, Daisuke Sugiyama, Motohiro Tamiya, Tatsuya Yoshida, Tetsunari Hase, Takao Yoshida, Kennosuke Karube, Yuka Maeda, Shohei Koyama, Hiroyoshi Nishikawa. Simultaneous activation of innate immune suppressive components impairs antitumor efficacy of immunotherapy against the innate immune system [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 2697.

Abstract 288: Middle-aged murine models of triple negative breast cancer (TNBC) show significant differences in tumor immune and stromal environments when compared to classic 6-8 week old models; Challenges and opportunities for preclinical efficacy studies

Abstract The immune system orchestra suffer a deficit of function during aging, and the incidence of the large majority of cancer types is significantly increased in elderly humans. Although BC can occur in young women, it is way more common in middle-age and elderly women. There is evidence that a tonic immune system is pivotal for the success of therapies aimed at awakening a dormant or exhausted immunological response against cancer cells by mean of checkpoint inhibitors (CI). However, rodent models of cancers are currently using very young mice of a few weeks of age, which have a fully functional and tonic immune system and thymus and thus might be poorly representative of adult human cancer patients. We have recently reported that that a triple therapy (TT) involving antigen-presenting cell activation by vinca alkaloids and the generation of new TCF1+ stem cell-like T cell (scT) clones by an alkylating agent can significantly improve the efficacy of the CI anti-PD-1 in models of cancer known to be otherwise CI-resistant such as TNBC and lymphoma (Falvo et al, Cancer Res 2021; Orecchioni et al, JCM 2023). TT effect was due to T cells, as it was abrogated by their in vivo depletion. In the present study we investigated differences in TNBC growth kinetics, TT preclinical activity and tumor microenvironment (TME, including intratumoral immune and stroma cells) in young (6-8w, representative of a 6y old human) versus adult (12m, representative of 40y humans) mice. Models included 4T1 and EMT6 TNBC cells, the former generating a mostly lymphoid TME and the latter generating a mostly myeloid TME. In both models, TT efficacy was similar in young and in adult mice, as the treatment abrogated TNBC local and metastatic growth. CD8+ (but not CD4+) scTs, likely crucial players in TT efficacy, were only slightly reduced in adult mice in spite of age-related thymus involution. This notwithstanding, single cell RNA (10x), IHC and flow cytometry analyses of immune (CD45+EPCAM-) and stroma (CD45-EPCAM-) cell populations indicated major differences in the TME of young vs adult mice. Adults had significantly less CD4+ scTs, B naïve and NK cells and significantly increased memory B cells. TME matrix CAF were skewed in young, while pro-inflammatory stromal populations and myofibroblasts were skewed in adults. Matrix CAFs down regulated a signature involved in different ECM-remodeling abilities, and up regulated metabolic and hypoxic pathways. Expression of genes encoding for glycoproteins, basement membrane components, and collagens were also upregulated. Our data indicate profound differences between young and adult mice immune and stromal TMEs that should be taken into consideration when selecting the age of mice to be used in preclinical efficacy studies in orthotopic immune competent mice. Citation Format: Paolo Falvo, Stephan Gruener, Stefania Orecchioni, Giovanna Talarico, Giulia Bravetti, Giulia Mitola, Federica Pisati, Iros Barozzi, Francesco Bertolini. Middle-aged murine models of triple negative breast cancer (TNBC) show significant differences in tumor immune and stromal environments when compared to classic 6-8 week old models; Challenges and opportunities for preclinical efficacy studies [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 288.

Abstract 4106: A novel synthetic long peptide vaccine composition targeting multiple PD-L1 T-cell epitopes exhibits anti-tumor efficacy in a syngeneic mouse colorectal cancer model

Abstract Background Cancer immunotherapy targeting immune checkpoints has shown promising clinical anti-tumor efficacy over the past decades. Monoclonal antibodies against PD-1 or PD-L1 have demonstrated clinical efficacy in a wide spectrum of cancer types. Despite the clinical success of therapies using immune checkpoint inhibitors (ICI), only a subset of patients shows long-lasting tumor remission, while resistance and toxicities to these agents remain to be addressed. This supports the need to explore alternative immunotherapeutic approaches or additional therapies for synergistic combinations to augment the anti-tumor efficacy for cancer patients receiving ICI therapies. Methods As an active immunotherapy approach, we developed a blend of four novel synthetic peptide vaccines each containing one PD-L1 T-cell epitope with individual covalent conjugation of the carrier protein, keyhole limpet haemocyanin (KLH), which is to break immune tolerance towards self-molecules. The four PD-L1 epitopes range from 30 to 40 amino acids in length, which target different domains of mouse PD-L1 protein. The PD-L1 peptides were synthesized using the technique of solid-phase peptide synthesis and purified by high performance liquid chromatography. All naked peptides were conjugated to KLH protein by thiol-maleimide reaction, followed by dialysis for purification. The peptide vaccines were formulated with a Toll-like receptor 9 agonist CpG ODN 1826 and aluminum hydroxide gel for animal immunization. Results These synthetic long PD-L1 peptide vaccines are highly immunogenic in a syngeneic BALB/c mouse model for a cell-mediated immune response when admixed and formulated with adjuvants. The vaccine composition is well tolerated by the mice with no evidence of toxicity and autoimmunity. In vivo administration of the vaccine formulation can elicit a positive PD-L1-specific T cell response in splenocytes with an IFN-γ ELISPOT assay. Moreover, in vivo vaccination of these PD-L1 peptide vaccines in a CT26 syngeneic mouse preventive colorectal cancer model exhibits a potent anti-tumor activity, with a 54% tumor growth inhibition (p<0.001) in tumor volume compared to that of the adjuvant-control group at the end of the study. Conclusions Taken together, our data demonstrate a proof-of-concept that T cell-based PD-L1 peptide vaccines are a promising therapeutic strategy for inducing anti-tumor immunity to combat cancers. Citation Format: Kenneth Nansheng Lin, Melvin Toh. A novel synthetic long peptide vaccine composition targeting multiple PD-L1 T-cell epitopes exhibits anti-tumor efficacy in a syngeneic mouse colorectal cancer model [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 4106.

Abstract 1559: IKZF1 plays a crucial role in the spread of melanoma

Abstract Melanoma is one of the most fatal type of skin cancer. If melanoma has spread to distant sites, it is refractory to existing therapies with the 5-year survival rate declining to 17%. Metastasis remains as the main barrier to successful therapy, persisting as the main cause of cancer-related death. Thus, understanding metastasis mechanism is vital. First, we confirmed that IKZF1 gene was more increased in circulating tumor cells than primary tumor cells. Using GFP+ B16F10, we generated an orthotopic melanoma mouse model and isolated GFP+ tumor cells of primary footpad, blood, and lung. In single cell RNA sequencing, IKZF1, IRF8, NFE2 genes were more upregulated in circulating tumor cells than primary tumor cells. We also found these correlations in human melanoma cell line orthotopic mouse model and Braf/Pten mouse model. Next, we conduct intervention study to find causality in IKZF1 and metastasis. IKZF1 KO led to fewer CTCs and IKZF1 inhibitor, lenalidomide, reduced metastasis in mice. Collectively, we demonstrate that IKZF1 could contribute the formation of CTC and IKZF1 could be a target for metastasis treatment. Citation Format: Jongwook Oh, Dong Ki Lee, Soyeon Lee, Hyun Woo Park, Heon Yung Gee. IKZF1 plays a crucial role in the spread of melanoma [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 1559.

Abstract 6334: Metabolic reprogramming enhances expansion and potency of CAR T cells

Abstract Cell therapies, such as Chimeric Antigen Receptor (CAR) T cell therapy, have revolutionized the treatment of hematological malignancies, but their use in solid tumors remains a significant challenge for the field. This is due, in part, to immunosuppressive mechanisms in the tumor microenvironment (TME), which include reduced oxygen tension, high interstitial pressure, and an abundance of immunosuppressive proteins, resulting in mitochondrial dysfunction, exhaustion, and depletion of adoptively transferred cells. Metabolic manipulation of therapeutic cells could overcome these barriers to enable cell therapy success in solid tumors. To this end, we have employed AVATAR technology (Xcellbio, San Francisco, CA), an incubation system that enables precise control of oxygen tension and hyperbaric pressure on cells in culture to improve the manufacture of adoptive T cell therapies. Using a CD19 CAR T model system, we have shown that transduction and expansion of CAR T cells under reduced oxygen and hyperbaric pressure conditions yield greater percentages and total numbers of lentiviral-transduced cells. While cells transduced in a standard CO2 incubator yielded 10-20% CD19 CAR+ cells, transduction in the AVATAR system with increased pressure yielded 15-40% CD19 CAR+ cells. Similarly, T cell cultures performed under pressurized AVATAR conditions generated ~2X more viable cells after 10 days. Furthermore, these metabolically reprogrammed cells exhibit enhanced potency, with improved anti-tumor cytotoxic activity in vitro. We have extended these studies to measure the ability of AVATAR-expanded CD19 CAR T cells to control the growth of CD19-expressing NALM6-Luc tumor cells in vivo compared to CAR T cells grown in a conventional CO2 incubator. Mice (female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratories, Bar Harbor, ME) dosed with the AVATAR-expanded cells exhibited good tumor control and increased persistence of CAR T cells in relevant organs, along with alterations in cell trafficking to the bone marrow and spleen. The transferred cells maintained a less differentiated phenotype, with central memory and effector memory populations, as measured by expression of the markers CD45RA and CD62L, dominating the T cell compartment. This work showcases the benefits of metabolic reprogramming to improve the yield and functional potency of CAR T cells, which has direct applications to reducing the cost and improving the efficacy of these lifesaving treatments. The AVATAR technology is currently being translated into a closed system bioreactor for use in GMP cell therapy manufacturing. Citation Format: Yewei Xing, Shannon Eaker, Yelena Bronevetsky, Candy Garcia, Hadia Lemar, Evan Massi, Albert Wong, Sheri Barnes, Derrik Germain, Scott Wise, James Lim. Metabolic reprogramming enhances expansion and potency of CAR T cells [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 6334.

Abstract 2060: Automation of 3D cancer spheroid assay for compound screening

Abstract Finding efficient drug combinations to treat cancer patients is critical for therapy success. Accordingly, there is a critical need to develop methods for efficient testing drug efficacy to discover new therapeutic targets. 3D cancer models are highly valuable tools for cancer research and drug development. However, the complexity of performing 3D assays remains a hurdle for adopting these methods for compound screening. To alleviate the bottlenecks that come with labor-intensive manual protocols we developed cell culture automation methods using CellXpress.ai instrument. The instrument enables automation of 2D or 3D culture for prolonged complex workflows. The CellXpress.ai provides automated plating, passaging, media exchanges, monitoring organoids, alone with compound treatment and the end point assays with high content image analysis. In the present study we describe full automation of imaging, analysis, and cell culture process which enables scaling up complex 3D cell-based assays and compound screening. As an example of 3D protocol, we automated culture and imaging of colorectal cancer 3D spheroids formed from HCT116 cell line in U-shape low attachment plates. HCT116 cells were expanded in 2D, then spheroids were formed after automated dispensing of cell suspension into U-shape 96 or 384 plates. After 48h spheroids were treated with 12 anti-cancer compounds, at multiple concentrations for 3-5 days, then stained and imaged. Cell plating, compound additions, media exchange, and staining were performed automatically by CellXpressAI instrument. During the culture spheroids were monitored using transmitted light, with immediate analysis of phenotypic changes, including inhibition of growth or spheroid disintegration. For endpoint assay spheroids were stained with combination of Hoechst nuclear stain and viability dyes Calcein AM and EtHD, then imaged, and analyzed for multiple read-outs including spheroid size, marker intensities, and live-dead cell scoring. We observed concentration-dependent inhibition of spheroid growth and cell death in response to anti-cancer compounds and evaluated effective compound concentrations for cytotoxic or cytostatic effects. Cell culture process automation for 3D assays, powered by imaging and image analysis has a potential to bring 3D biology into another level, allowing to increase throughput and reproducibility, and enabling variety of high throughput drug discovery and precision medicine applications. Citation Format: Oksana Sirenko, Angeline Lim, Astrid Michlmayr, Zhisong Tong, Emilie Keidel, Felix Spira. Automation of 3D cancer spheroid assay for compound screening [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 2060.

Abstract 57: Ibrutinib improves chimeric antigen receptor T cell control of leukemia by inhibiting myeloid-derived suppressor cells

Abstract Background: In patients with chronic lymphocytic leukemia (CLL), we and others have shown that the addition of the BTK inhibitor ibrutinib to CART cells increases the rates of durable complete responses. The mechanism for this remains unknown. Lymphodepletion (LD) is considered critical for CART cell therapy success, yet emergency myelopoiesis during recovery from LD may induce a surge of myeloid-derived suppressor cells (MDSC), particularly in patients with advanced cancer. BTK inhibition has been shown to inhibit MDSC function in vitro. We therefore hypothesized that administration of ibrutinib during and following LD could reduce MDSC-mediated suppression of CART cells and thereby improve CART cell outcomes. Methods: To explore the effects of ibrutinib on human MDSCs, we engineered suppressive monocytes through in vitro culture of CD14+ cells in the presence of IL-6 and GM-CSF. We added ibrutinib to the culture on Days 0-4 and compared the ability of the monocytes to inhibit proliferation of human CAR-T cells. To model CLL treatment with CART in immunocompetent mice, we used the Em-TCL1 adaptive transfer model of CLL. Mice were treated with ibrutinib (I), cyclophosphamide and fludarabine (FC), or ibrutinib + FC (FC+I). MDSCs were isolated from these mice for in vitro function studies. In subsequent experiments mice were also treated with syngeneic CART19 with or without FC or FC+I. Results: In vitro generated human MDSC suppressed CART19 function, and this was prevented by pre-treatment of MDSC with ibrutinib. Myeloid cells from the spleens of CLL-bearing mice inhibited syngeneic CART19 function ex vivo regardless of treatment with FC or I alone, but myeloid cells harvested from CLL-bearing mice treated with FC+I lost suppressive function. Finally, mice treated with CART19 after a LD regimen resembling that in our recently reported clinical trial (FC+I) showed a survival advantage over all other groups. Conclusions: BTK inhibition with ibrutinib prevents myeloid cell suppression of CART cells in vitro in human and murine systems. When combined with lymphodepleting chemotherapy (FC), ibrutinib enhances CART cell control of CLL in vivo, possibly by preventing MDSC-mediated suppression of CART cells. Citation Format: Benjamin F. Frost, Olga Shestova, Feng Shen, Chia Sharpe, John C. Byrd, Saar I. Gill. Ibrutinib improves chimeric antigen receptor T cell control of leukemia by inhibiting myeloid-derived suppressor cells [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 57.

Current patient care of systematic lupus erythematosus in Rhineland-Palatinate and Saarland

Systemic lupus erythematosus (SLE) is aclinically heterogeneous autoimmune disease that is associated with great suffering for those affected, as well as high socioeconomic costs. Early diagnosis and adequate medical care are essential for amild course of the disease. However, there is a lack of current figures and data on the care situation of patients in the area. Atotal of 1546 general practitioners, rheumatologists, neurologists, nephrologists and dermatologists in Rhineland-Palatinate and Saarland were interviewed by fax or mail using aquestionnaire regarding epidemiology, symptoms, therapy and therapy success. In addition, there was the possibility of making suggestions for improvement. Five out of six of the 635 reported SLE patients were female. The most common main symptoms were arthralgia, fatigue, myalgia, and skin changes. Of the patients, 68% received antimalarials (AMs), whereas 46% were treated with glucocorticoids (GCs) and 50% with an immunosuppressant (IS), mainly methotrexate. In terms of comorbidities, patients suffered mainly from cardiovascular disease, fibromyalgia syndrome and depression. Rheumatologists also frequently described anaemia, diabetes mellitus and osteoporosis. Compared with guideline recommendations, the low rate of AMs in therapy was particularly striking in patients not treated by rheumatologists (35% on average compared with 81% for rheumatologists). Additionally, (sustained) high doses of GCs are not in line with literature recommendations. In the free text field, the main requests were for more rheumatologists in private practice and faster appointment scheduling, as well as better communication and networking. In addition, the desire for more training and education was frequently expressed..

“Pap smear horror stories, self loathing, feeling like I was less of a woman”: A qualitative content analysis of an online vaginismus forum

Objective: To understand what concerns and experiences people with vaginismus share online anonymously but may not share with clinicians. Methods: Two researchers compiled all comments and posts made from November 2019 to April 2020 within an online vaginismus forum ( n = 311) for content analysis. Two researchers separately performed inductive coding, categorized quotes into overarching categories and codes, and resolved inconsistencies with a third-party adjudicator. Results: Our analysis highlighted three key categories: “psychosocial concerns” (34%), “discussion of pain” (33%), and “lack of access to vulvovaginal healthcare” (34%). The category “psychosocial concerns” comprised of the codes “shame and/or social isolation” (12%), “mental health comorbidities” (9%), “fear of partner separation” (7%), “cultural/religious stigma” (2%), “partner separation” (2%), and “fear of infertility or vaginal delivery” (1%). The category “discussion of pain” comprised of the codes “successful therapies” (16%), “sexual pain” (9%), “nonsexual pain” (6%), and “unsuccessful therapies” (2%). The category “lack of access to vulvovaginal healthcare” comprised of the codes “lack of standardized therapy guidelines” (8%), “self diagnosis” (7%), “fertility testing and/or treatments limited by vaginismus” (5%), “clinicians dismissing symptoms” (4%), “inadequate clinician knowledge about vaginismus” (2%), “inadequate health and sex education in schools” (2%), “geographical barriers” (2%), and “COVID-19” (2%). Conclusion: This novel qualitative study of an online vaginismus forum highlights the need for a patient-centered approach to inform future research, medical education, and clinical practice. Our findings suggest we reevaluate the standard of care for patients with vaginismus and expand on existing knowledge of the impact of sexual health stigma on social and health outcomes.

Evaluating immunotherapeutic outcomes in triple-negative breast cancer with a cholesterol radiotracer in mice.

Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement for cholesterol in the activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in preclinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and -nonresponsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells allows detection of T cell activation and has potential to assess the success of ICI therapy.

Chimeric Antigen Receptor (CAR)-T Cell Therapy for Non-Hodgkin's Lymphoma.

This review focuses on the use of chimeric antigen receptor (CAR)-T cell therapy to treat non-Hodgkin's lymphoma (NHL), a classification of heterogeneous malignant neoplasms of the lymphoid tissue. Despite various conventional and multidrug chemotherapies, the poor prognosis for NHL patients remains and has prompted the utilization of groundbreaking personalized therapies such as CAR-T cells. CAR-T cells are T cells engineered to express a CAR that enables T cells to specifically lyse tumor cells with extracellular expression of a tumor antigen of choice. A CAR is composed of an extracellular antibody fragment or target protein binding domain that is conjugated to activating intracellular signaling motifs common to T cells. In general, CAR-T cell therapies for NHL are designed to recognize cellular markers ubiquitously expressed on B cells such as CD19+, CD20+, and CD22+. Clinical trials using CAR-T cells such as ZUMA-7 and TRANSFORM demonstrated promising results compared to standard of care and ultimately led to FDA approval for the treatment of relapsed/refractory NHL. Despite the success of CAR-T therapy for NHL, challenges include adverse side effects as well as extrinsic and intrinsic mechanisms of tumor resistance that lead to suboptimal outcomes. Overall, CAR-T cell therapies have improved clinical outcomes in NHL patients and generated optimism around their future applications.

Proinflammatory polarization of engineered heat-inducible macrophages reprogram the tumor immune microenvironment during cancer immunotherapy

The success of macrophage-based adoptive cell therapy is largely constrained by poor polarization from alternatively activated (M2-like) to classically activated (M1-like) phenotype in the immunosuppressive tumor microenvironment (TME). Here, we show that the engineered macrophage (eMac) with a heat-inducible genetic switch can induce both self-polarization of adoptively transferred eMac and re-polarization of tumour-associated macrophages in response to mild temperature elevation in a mouse model. The locoregional production of proinflammatory cytokines by eMac in the TME dose not only induces the strong polarization of macrophages into a classically activated phenotype, but also ensures that the side effects typical for systemically administrate proinflammatory cytokines are avoided. We also present a wearable warming device which is adaptable for human patients and can be remotely controlled by a smartphone. In summary, our work represents a safe and efficient adoptive transfer immunotherapy method with potential for human translation.

Evaluation of a scoring system to predict treatment success with single-dose methotrexate in ectopic pregnancy.

This study sought to assess the efficacy of a newly developed scoring system in predicting treatment outcomes for ectopic pregnancy among patients undergoing single-dose methotrexate therapy. The primary research question centered on the reliability and predictive accuracy of objective parameters in determining methotrexate therapy success. Conducted as a retrospective single-center cohort study, data from 172 ectopic pregnancy patients treated with methotrexate between January 2021 and January 2023 were analyzed. Parameters including adnexal mass size, peritoneal fluid presence, yolk sac identification, endometrial thickness, ectopic pregnancy location, and initial B-hCG levels were meticulously evaluated for their association with treatment outcomes. Following the exclusion of 21 emergency surgery cases, the final analysis comprised 151 patients. Notable associations were observed between specific parameters (fetal cardiac activity, adnexal mass size > 3.5cm, peritoneal fluid presence, yolk sac identification, endometrial thickness > 10mm, and initial B-hCG levels) and treatment outcomes (p < 0.001). Additionally, the novel scoring system demonstrated promising predictive performance. At a cutoff of 2.50, it achieved a sensitivity of 91.7% and a specificity of 59.7%. Increasing the cutoff to 3.50 resulted in a sensitivity of 94.0%, with a specificity of 46.3%. Objective parameters, particularly those integrated into the developed scoring system, exhibited substantial associations with methotrexate therapy outcomes in ectopic pregnancy. These findings underscore the potential of an objective scoring model to significantly influence clinical decision-making in therapy, offering avenues for enhanced prognostication and patient care in treatment outcomes.

Success of Lesion Sterilization and Tissue Repair Therapy and Pulpectomy in the Management of Infected Primary Molars with Poor Prognosis.

To establish lesion sterilization and tissue repair (LSTR) therapy as an alternate treatment option in managing infected primary molars with poor prognosis that were indicated for extraction, thereby fulfilling the objective of retaining the primary tooth till its normal exfoliation in the dental arch. A total of 84 children who met the inclusion criteria requiring extraction in 142 teeth involving primary molars were included in the study. The selected patients were allocated to two groups, that is, group I-LSTR therapy with 3Mix-MP paste and group II-pulpectomy with metapex. All the treated teeth were then clinically and radiographically evaluated after 1, 3, 6, 9, and 12 months, respectively, to determine the success between groups I and II. Pearson's Chi-squared test along with the z-test was used to compare the clinical and radiographic success of the two groups (p < 0.05). Pain and tenderness were completely resolved within one month of follow-up in both groups. Abscesses were resolved completely at 1 month in the pulpectomy group and mobility was resolved at 6 months follow-up in both groups. Interradicular and periradicular radiolucency persisted even at 12 months of the follow-up period in both groups. The intergroup comparison revealed no statistical differences between LSTR and pulpectomy procedure and both were equally effective at all time intervals (p > 0.05). Both LSTR therapy with 3Mix-MP and pulpectomy with metapex showed 100% clinical success rates. Radiographically no changes were observed even at the 12-month follow-up period in both groups. LSTR therapy can be an alternative treatment option for pulpally involved primary teeth with poor prognosis and in cases where mechanical instrumentation could not be achieved due to physiologic root resorption. Sefa I, Garg N, Pathivada L, et al. Success of Lesion Sterilization and Tissue Repair Therapy and Pulpectomy in the Management of Infected Primary Molars with Poor Prognosis. Int J Clin Pediatr Dent 2024;17(1):41-47.