Changes In Microenvironment Research Articles

Abstract 1174: Incorporation of multisector analysis into the design of personalized DNA vaccines for patients with newly diagnosed glioblastoma

Abstract Glioblastoma (GBM) is the most common malignant central nervous system (CNS) tumor in adults. Despite multimodality treatment including surgery, radiation, and chemotherapy, patients with GBM have a median survival of less than 2 years. Recent clinical trials have reported promising results using cancer vaccines to stimulate a tumor-directed immune response in several histologies. The majority of trials targeted tumor neoantigens derived from a single tumor sample, which limits the antigen pool in spatially heterogeneous cancers like GBM. Our group previously implemented multisector sequencing into the design of personalized peptide vaccines to increase the candidate pool of targetable neoantigens (NCT03422094). Although the peptide vaccine treatment was successful in stimulating an effector T cell response, it was limited by a long turnaround time from vaccine design to administration and a low peptide production rate. Therefore, we incorporated multisector sampling into the design of a personalized DNA-based GBM vaccine (NCT04015700). An average of 33% of the candidate targetable neoantigens were spatially restricted to a single sampled region, which would have been missed without multisector sequencing. DNA-based vaccines are potentially advantageous over peptide-based vaccines because they enable a higher neoantigen payload and faster manufacturing time with potent immunogenicity. Here, we report the results from 9 subjects enrolled onto the study. Spatially distinct tumor regions were subjected to whole exome sequencing (WES) and RNA-sequencing (RNA-seq), data from which were used to identify neoantigens through the pVACseq algorithm (http://pvactools.org). An average of 32 neoantigens were included in each DNA vaccine, compared to an average of only 9 for the peptide vaccine, and the turnaround time from date of surgery to administration of the DNA vaccine was about half the time. Three subjects had radiographic evidence of tumor progression prior to vaccination, highlighting the aggressive nature of GBM and the need for short manufacturing timeframes. PBMCs pre- and post-DNA vaccination were collected from 7/9 subjects. All subjects presented detectable responses and 6/7 tested revealed a sustained increment in T cell reactivity against tumor neoantigens post-treatment by IFN-γ ELISpot. Intracellular staining showed a neoantigen-specific antitumor CD8/CD4 T cell cytolytic (CD69+, CD107a+) and proliferative (Ki67+) profile. Post-vaccination tumor resections were performed for 2 subjects who also were treated with PD1 blockade upon progression, providing an opportunity to explore vaccine-induced changes in the tumor microenvironment. Herein, we demonstrate the advantages of incorporating multisector sequencing into the development of DNA-based GBM vaccines and provide insights into the resulting immune responses. Citation Format: Elizabeth A. Garfinkle, Katherine E. Miller, Alexandra J. Livingstone, Renzo Perales-Linares, Neil Cooch, Alfredo Perales-Puchalt, Sarah Rochestie, Joann Peters, Niranjan Y. Sardesai, William E. Gillanders, Elaine R. Mardis, Gavin P. Dunn, Tanner M. Johanns. Incorporation of multisector analysis into the design of personalized DNA vaccines for patients with newly diagnosed glioblastoma [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 1174.

Abstract 3858: Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS)

Abstract Introduction: NeoICB is a promising approach for pts with DDLPS and UPS. However, biomarkers to identify which pts benefit from this treatment and understanding the mechanisms of response and resistance to ICB remains a challenge. Methods: We conducted a randomized phase 2 trial of neoICB (anti-PD1 +/- CTLA4) in pts with resectable DDLPS (n=17) and UPS (n=10). Pts with UPS received concurrent neoICB and radiation. The primary endpoint of the trial was pathological response; 3 DDLPS and 9 UPS pts were responders (R) and 14 DDLPS and 1 UPS pts were non-responders (NR). Peripheral blood mononuclear cells (PBMCs) were collected at baseline, prior to cycle 2 of neoICB (C2D1), and at the time of surgery (SURG). We performed mass cytometry (CyTOF) to identify immune cells associated with response and their dynamic changes with neoICB. We designed a custom 41-parameter panel using CD45 live cell barcoding with 31 markers for immune profiling, of which 23 were selected to detect key cell lineage and 8 to detect signaling markers. Batch correction and high dimensional data analyses were performed using CyCombine and CyTOF workflow R packages. Longitudinal comparisons between immune cell populations were conducted using Mann-Whitney U Test. Results: Of 79 available PBMC samples from 27 pts, 63 passed quality control after thawing (≥30% viability) and data clean-up (at least 10,000 events). Data was down-sampled to 5000 events for analysis. Of 19 immune cell types identified, monocytes were the most common followed by CD4+ and CD8+ T cells. There was an early increase in CD38+ effector/effector memory (EM) T cells at C2D1 in pts with DDLPS (CD8+ p=0.005, CD4+ p<0.001) and a similar increase observed in pts with UPS (CD8+ C2D1 p=NS, SURG p=0.034; CD4+ C2D1 p=0.024, SURG p=0.06) and these cells had higher Ki67 expression than CD38- T cells (p<0.005). At baseline, R pts with DDLPS had higher fractions of CD38+ EM CD8+ T cells than NR pts (p=0.044). Analysis in the UPS cohort was limited by the low number of NR pts. Higher expression of TIGIT was observed on CD38+ EM CD8+ T cells compared to CD38- counterparts at all time points. Conclusion: Peripheral CD38+ EM CD8+ T cells are associated with response to neoICB in pts with DDLPS. Longitudinal peripheral immune profiling reveals an increase in CD38+ EM CD8+ and CD4+ T cells with neoICB in pts with DDLPS and UPS. These cells are proliferative and express higher levels of TIGIT, which could be a future target for therapeutic exploration. Importantly, changes in the peripheral immune populations may precede changes in the tumor microenvironment (TME) as preliminary analyses of the TME did not identify early (C2D1) changes. TME imaging mass cytometry analyses are currently underway to associate peripheral and intratumor changes in immune populations. Citation Format: Elise F. Nassif, Hsinyi Lu, Michael Stanford, Karen Millerchip, Diana Romero, Jared Malke, Brenna Matejka, Jeffrey E. Gershenwald, Susmita Kumari, Duncan H. Mak, Angelique J. Lin, Jared K. Burks, Wei-Lien Wang, Alexander J. Lazar, Neeta Somaiah, Cara Haymaker, Christina L. Roland, Emily Z. Keung. Peripheral CD38+ effector memory (EM) T cells are dynamic biomarkers of response to neoadjuvant immune checkpoint blockade (NeoICB) in patients (pts) with resectable dedifferentiated liposarcoma (DDLPS) and undifferentiated pleomorphic sarcoma (UPS) [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 3858.

Abstract 5285: DKK1 blockade inhibits progression of bone metastases via multiple mechanisms and synergize with zoledronate treatment

Abstract Background: Despite the important breakthroughs of immune checkpoint blockade (ICB) therapy in multiple primary cancers, responses of ICB therapy in bone metastases remain extremely poor, largely due to the highly immunosuppressive bone metastasis microenvironment. However, the current understanding of bone metastasis microenvironment is still incomplete, and specific treatment targets for patients with bone metastasis are still lacking. Methods: Transcriptome sequencing data of gastric and breast cancer bone metastasis samples from The TCGA and GEO databases were obtained. The correlation between DKK1 expression and prognosis, intertumoral immune infiltration of bone-metastatic patients was analyzed. Mouse bone metastasis models from gastric, breast and lung cancer were established, and in vivo treatment efficacy of DKK1 and/or zoledronic acid was evaluated by micro-computed tomography (CT), micro-magnetic resonance imaging (MRI) and immunohistochemistry (IHC) staining. The changes of immune components wthin bone-tumor microenvironment were explored via flow cytometry, CyTOF and scRNA-seq analysis. The impact of DKK1 on different immune cell types was analyzed via in vitro co-culture models. Results: Serum detection of gastric cancer patients from our center showed that compared with non-metastatic patients, DKK1 concentration in patients with bone metastasis was the significantly increased (p<0.0001); and compared with other metastatic sites of breast cancer patients, bone metastases had the highest level of DKK1 mRNA expression (p<0.05). Using multiple bone metastasis mouse models, we found that DKK1 blockade significantly controlled the tumor burden of bone metastases. Results of micro-CT and TRAP staining showed that the bone destruction and osteoclast activity within bone lesions were significantly inhibited after DKK1 blockade. In addition, results of flow cytometry and immunofluorescence staining on bone metastasis samples showed that both innate and adaptive anti-immune responses were obviously improved after DKK1 blockade, including increased infiltration of CD8+ T cells, M1 macrophages and decreased infiltration of M2 macrophages. Mechanistically, DKK1 directly induced inhibition of CD8+ T cell activation, M2 polarization of macrophages and maturation of osteoclasts, which all contributes to the immune suppression in the bone metastasis microenvironment. Finally, combined therapy of DKK1 blockade and zoledronic acid had synergistic treatment effects in bone metastases. Conclusion: Our study provides novel insights into the multiple role of DKK1 in the bone metastasis immune microenvironment. DKK1 is a potential immunotherapeutic target for bone metastases, and dual blockade of DKK1 and zoledronic acid is a promising combination immunotherapy strategy for patients with bone metastasis. Citation Format: Tao Shi, Kaijie Liang, Yipeng Zhang, Xiaoyu Zhou, Baorui Liu, Jia Wei. DKK1 blockade inhibits progression of bone metastases via multiple mechanisms and synergize with zoledronate treatment [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 5285.

Abstract 1502: Deciphering the spatial and temporal dynamics of premetastatic niche in lung mediated by Treg and myeloid cell interactions

Abstract Introduction: The lung is a frequent site for metastases, where pre-metastatic niches (PMN) form in anticipation of tumor cell arrival. A deeper understanding of the molecular and immune features underlying the PMN formation may facilitate developing innovative strategies to prevent and treat metastasis more effectively in its early stages. Methods: In this study, we developed mouse models by subcutaneously implanting the 344SQ cell line, derived from the metastasis of a genetically engineered mouse model (GEMM) harboring KrasG12D/+; p53R172H/+ mutations, which reliably metastasizes to the lungs. We collected paired samples of the subcutaneous tumors, peripheral blood mononuclear cells (PBMCs), and lung tissue at pre-metastatic, early-metastatic, and late-metastatic phases. These samples were subjected to single-cell RNA sequencing (scRNA-seq), single-cell TCR/BCR sequencing (scTCR/BCR-seq), and spatial gene expression profiling with 10X Genomics Visium technology. Results: ScRNA analysis revealed large-scale comprehensive longitudinal changes in cell components throughout the invasion and metastasis cascade, which were validated by spatial gene expression. Furthermore, analysis of Visium data deduces the spatial information of these dynamic changes.We identified a subset of tumor-associated Tregs (TA_Tregs, enriched in the subcutaneous tumor tissues) that existed in small amounts in the lung tissue in the pre-metastatic phase when there are no tumor cells detectable in the lung. These cells were characterized by high expression of Treg-associated suppressive genes (Tnfrsf9, Tnfrsf4, Tnfrsf18, Traf1, Nfkbia), inhibitory molecules (Ikzf2, Il2ra, Tight, Pdcd1), cytokines and chemokines (Ccr5, Ccr8, Ccl5, Cxcr6) and other Treg effector molecules (Id2, Hif1a, Rora, Il1r2, Itgav, Stat1, Icos, Areg). The scTCR-seq results and trajectory analysis revealed the TA_Tregs might derive from minor clones of lung resident Tregs (LR_Tregs) and play an active role in remodeling an immunosuppressive microenvironment by closely interacting with other cell components in the TME. We also observed a significant alteration in the ratio of macrophage subpopulations between Macro_Spic and Macro_Vcan. This ratio exhibited a continuous decrease in lung tissues, which could be an early indication of the initiation of pre-metastatic niche (PMN) conditioning. Conclusion: By simultaneously characterizing cellular gene expression, immune repertoires, and their corresponding spatial information, we revealed the dynamic changes of the lung tissue microenvironment (LTME) during from a normal state to pro-metastasis state at single-cell resolution. The LTME of metastatic organs may be primed before the establishment of metastasis. Citation Format: Yanhua Tian, Jian-Rong Li, Bo Zhu, Jared Fradette, Hong Chen, Chenyang Li, Leticia Rodriguez, Daniel Kraushaar, Luisa Solis, Alexandre Reuben, Ignacio Wistuba, Don L. Gibbons, Chao Cheng, Jianjun Zhang. Deciphering the spatial and temporal dynamics of premetastatic niche in lung mediated by Treg and myeloid cell interactions [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 1502.

Abstract 5559: The anti-tumor efficacy and immune effects of combining of ceralasertib, an oral ATR kinase inhibitor, with imipridones, in prostate cancer treatment in vitro and in vivo

Abstract Prostate cancer is the most common cancer and the second leading cause of cancer death among men in the United States. There is an estimated 10% to 50% of cases progress to metastatic castration resistant prostate cancer (mCRPC) state within 3 years of diagnosis. mCPRC remains lethal despite therapeutic advances. There is approximately 20% of mCRPC patients present somatic DNA damage repair (DDR) gene mutations. Ceralasertib, formerly known as AZD6738, is a potent and selective orally bioavailable inhibitor of the ataxia tenlangiectasia and Rad3-related (ATR) kinase, which is involved in DNA repair in response to DNA damage and replication stress. Preclinical studies have demonstrated ATRi sensitizing alterations in DNA damage response (DDR) genes. Ceralasertib’s antitumor activity as a monotherapy in treating prostate cancer is moderate. Thus, we combined ceralasertib with imipridones (ONC201 and ONC206), which target mitochondrial caseinolytic protease P (ClpP) and the integrated stress response, resulting in enhanced antitumor efficacy in vitro. Prior data have demonstrated sensitivity of 4 prostate cancer lines to ceralasertib and imipridones monotherapies, synergistic activities with the combination treatments, and immune enhancement effects with the combination treatments when co-cultured with NK92MI cell line. We proceeded to a short term in-vivo study validating the combination treatment efficacy. Preliminary results include cytokine profiling using the Luminex 200 technology to understand treatment induced changes in the tumor microenvironment (TME) from both in vitro and in vivo studies. Our results guide novel clinical trials for effective clinical responses in mCPRC patients. Citation Format: Yutong Linda Xia, Leiqing Zhang, Maryam Ghandali, Maximilian P. Schwermann, Wafik El-Deiry. The anti-tumor efficacy and immune effects of combining of ceralasertib, an oral ATR kinase inhibitor, with imipridones, in prostate cancer treatment in vitro and 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 5559.

Abstract 110: Adoptive transfer of tumor antigen-specific T cells alters the renal tumor microenvironment in the presence and absence of immunotherapy

Abstract Immunotherapies are the standard of care for patients with renal cancer, yet their effects on renal tumor antigen-specific CD8 T cell responses and their subsequent ability to modulate the renal tumor microenvironment remain difficult to evaluate. In order to address this gap in the field, we developed a novel murine model to examine tumor-antigen specific CD8 T cell immune responses in orthotopic renal cancer utilizing a new cell line, Renca-tERK-LUC. Parental Renca cells were transduced with a lentivirus expressing the naturally-occurring, mutated tERK peptide found in CMS5 fibrosarcoma cells to create Renca-tERK-LUC tumor cells. The tERK mutation consists of a one base pair change that creates an Sfc1 site, so it can be detected by RT-PCR followed by restriction enzyme digestion. Our data show that Renca-tERK-LUC cells express tERK at high levels and grow progressively in control BALB/c mice, but are rejected in DUC Thy1.1 mice, which contain endogenous TCR transgenic CD8+ T cells specific for tERK peptide/MHC I complexes. We then performed adoptive transfers of 5 × 105 DUC Thy1.1 T cells into mice with established, orthotopic Renca-tERK-LUC tumors to evaluate: 1) the effects of immunotherapy on tumor-antigen-specific T cell responses; and 2) the effects of tumor-antigen-specific T cells on the broader renal tumor microenvironment. Our data indicate that the transfer of relatively low numbers of tumor antigen-specific CD8+ T cells induces transcriptomic and cellular changes in the tumor microenvironment in both the presence and absence of immunotherapy. Defining the parameters surrounding tumor-antigen-specific T cell responses and their ability to modulate the renal tumor microenvironment under the influence of clinically relevant immunotherapeutic intervention has the potential to increase the understanding of immunity to renal tumors, which could ultimately prove beneficial for renal cancer patients. Citation Format: Holly Stephens, Elizabeth Elkins, Francesca Dempsey, Zoey Swalley, Jing Li, Jianqing Zhang, Richard Kirkman, Bo-Ruei Chen, Zachary Roberts, Sunil Sudarshan, Barry Sleckman, Hubert Tse, Daniel L. Smith, Lyse A. Norian. Adoptive transfer of tumor antigen-specific T cells alters the renal tumor microenvironment in the presence and absence of 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 110.

Abstract 2939: Harnessing in vivo CRISPR screen to uncover metabolic dependencies during cancer progression

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with the worst prognosis and limited treatment options. TNBC cells undergo metabolic reprogramming in response to nutrient limitations and changes in the tumor microenvironment throughout cancer progression. In the early stages, tumor cells are found to prioritize nutrient uptake and biosynthesis, but as they grow, intensified competition for nutrients fosters the emergence of subpopulations of cancer cells that excel in utilizing specific nutrients. The substantial metabolic pathways and genes that regulate each phase of cancer progression remain poorly understood. To address this, we have developed a stable integration, site-specific, and inducible CRISPR-Cas9 system that allows for temporal control of genetic perturbation. We conducted an unbiased in vivo loss-of-function CRISPR screen aimed at identifying metabolic vulnerabilities at different phases of cancer progression of TNBC. The inducible CRISPR system was activated only at the desired study period of each phase, thus capable of uncovering metabolic dependencies for TNBC proliferation at each distinct phase of cancer progression. As many large-scale CRISPR screens have previously been performed to identify gene dependencies for cancer progression in vitro, our CRISPR screen could address the shortcomings of in vitro screens by identifying novel essential metabolic genes that are significantly important for cancer growth in vivo. Preliminary data from our CRISPR screen revealed different pathways to be significantly important for tumor growth and proliferation at different phases of TNBC progression. For example, we find that ferroptosis is altered during the early lag phase of TNBC cancer progression, whereas, at the later stage of cancer progression, metastatic tumors upregulate serine metabolism, suggesting that secondary tumors could have distinct metabolism from the primary sites. Altogether, the findings from this study could provide valuable insights into stepwise metabolic dependencies arising from microenvironmental limitations and identify potential therapeutic targets at each phase of cancer progression. Moreover, our CRISPR-Cas9 model holds promise for dissecting the complex mechanisms of other cancer progressions. Citation Format: Seow Qi Ng, Zhengwei Wu, Jessica A. Lidster, Wai Leong Tam. Harnessing in vivo CRISPR screen to uncover metabolic dependencies during cancer progression [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 2939.

Abstract 6660: The novel codon-modified Zika virus CDX-602 shows anti-tumor efficacy and remodels the tumor microenvironment in preclinical models

Abstract Multiple oncolytic viruses have been shown to induce multifaceted changes in the tumor microenvironment, that ultimately support induction of anti-tumor immune responses. We have previously demonstrated that CodaLytic, a codon-modified influenza virus, can kickstart the cancer immunity cycle at various steps in several mouse models with differing baseline immune contextures. Here, we are describing the activity of a new codon-modified virotherapeutic derived from the same synthetic viral engineering platform [Coleman et al., Science 2008]. Zika virus strain MR766 was synthetically codon-modified at 664 positions in the envelop gene to yield CDX-602. Virus was originally passaged in Vero cells and working stocks were expanded in BSR-T7 cells for in vivo use after titration on MA104.1 cells. Efficacy after intratumoral injection of 107 PFU/dose CDX-602 alone or in combination with 200 μg/dose αPD-1 blockade (RMP1-14, i.p.) was determined in subcutaneous B16-F10 melanoma and orthotopic EMT6 breast cancer models (n = 10). Changes in the tumor immune infiltrate were characterized using flow cytometry 6 or 8 days after treatment initiation, respectively (n = 5). In the immunotherapy-resistant B16-F10 model, CDX-602 monotherapy led to highly reproducible tumor growth inhibition (TGI, 52%, p < 0.0001 vs vehicle control), resulting in modest, but significant prolongation of median survival (25%, p < 0.01). Anti-tumor efficacy correlated with increased tumor immune infiltration with CD4+ and CD8+ T cells (R2 = 0.68 and 0.67, respectively, p < 0.01). Importantly, frequencies of cross-presenting dendritic cells (cDC) were increased 3.8-fold (p < 0.0001) after virus treatment, while no compensatory influx of CD206+ macrophages was observed, suggesting immune contexture changes conducive to anti-tumor immune induction. Efficacy was confirmed in the EMT6 model, in which monotherapy TGI was 44% and thus on par with αPD-1 blockade (50%, both p < 0.0001 vs control). Combination viroimmunotherapy in this model further improved efficacy, leading to 71% TGI and a doubling in complete regressions to 40%. Inclusion of CDX-602 in the treatment regimen led to similar changes in immune infiltration, in particular recruitment of CD8+ T cells and cDCs (9.7-fold and 6.9-fold increase after combination, respectively). Additional ex vivo analyses to confirm induction of anti-tumor immune responses are ongoing. Taken together, this preclinical data establishes CDX-602 as a novel virotherapeutic candidate that engages multiple mechanisms of action that contribute to anti-tumor activity. CDX-602 is the second virotherapeutic candidate derived from Codagenix’s codon modification platform, further supporting the utility of this synthetic viral engineering technology for viroimmunotherapy of cancer and highlighting its potential across multiple viral species. Citation Format: Nusrat Jahan, Yiwen Zhao, Katarina Blagovic, Ying Wang, J Robert Coleman, Steffen Mueller, Johanna K. Kaufmann. The novel codon-modified Zika virus CDX-602 shows anti-tumor efficacy and remodels the tumor microenvironment in preclinical models [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 6660.

Abstract 5924: RNAi mediated inhibition of beta-catenin demonstrates anti-tumor efficacy and immune microenvironment modulation in preclinical hepatocellular carcinoma models

Abstract Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide, and the third most common cause of cancer-related deaths. For the 40% to 50% of HCC patients who are candidates for systemic therapies, the landscape has evolved significantly in the last 10 years, with most recently, immunotherapies such as combination of atezolizumab and bevacizumab emerging as the current first-line systemic therapy for patients with intermediate or advanced disease. However, there are no predictive biomarkers for patients with HCC to enable selection of those most likely to benefit from these therapies and this remains a disease with high unmet medical need. Activation of the WNT pathway via mutational events or indirect pathway activation is implicated in many cancers including up to 50% of HCC patient tumors. High expression of beta-catenin, encoded by the CTNNB1 gene, has been shown to correlate with decreased survival and more rapid disease progression in HCC patients. In addition, WNT/beta-catenin pathway activation has been shown to result in immune exclusion and resistance to immunotherapy in HCC. ALN-BCAT comprises a chemically modified siRNA encapsulated in a lipid nanoparticle formulation which generates robust and highly specific reductions of CTNNB1 mRNA, downstream biomarkers and tumor cell proliferation in in vitro model systems. Anti-tumor activity has been demonstrated in multiple in vivo orthotopic HCC mouse models, in both early and late treatment settings as measured by liver to body weight ratio. Reductions in tumor burden were orthogonally confirmed by histology analysis and shown to be associated with changes in CTNNB1-related biomarkers. Finally, ALN-BCAT treatment results in changes in the tumor microenvironment in a mouse syngeneic HCC model, with increases in CD4+ and CD8+ T cells and reductions in M2 macrophages, consistent with a shift to a more immune stimulatory anti-tumor microenvironment. Here, we demonstrate that ALN-BCAT provides a novel approach to the inhibition of the WNT pathway as a therapeutic approach for treatment of HCC patients and WNT pathway activated tumors. Citation Format: Tulin Dadali, Spencer Miller, Gaurav Saawant, Stephen Abbott, Mikyung Yu, Svetlana Morskaya, Brandon Lehrich, Satdarshan Monga, Gloria Lau, Martin Maier, Wendy Broom. RNAi mediated inhibition of beta-catenin demonstrates anti-tumor efficacy and immune microenvironment modulation in preclinical hepatocellular carcinoma models [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 5924.

Abstract 4884: Evaluating the utility of in silico variant annotation tools for cancer driver detection

Abstract In cancer genomics, precise variant annotation is crucial for clinical decisions, drug development, and research. The burgeoning genomic data offers an opportunity to use data-driven approaches to generate knowledge that supports clinical decisions. Particularly machine learning (ML) and Deep learning (DL), are becoming essential, as their application is fast, scalable, simple to implement, and generates reproducible results. The methods ranging from simple sequence-based alignment scoring to advanced algorithms like Logistic regression, Support vector machine, and Recurrent neural networks, have been employed by multiple variant annotation tools. This study compares in-silico methods available in Ensembl's Variant Effect Predictor (VEP) using a test dataset with COSMIC annotations. ML/DL success relies on robust training sets with comprehensive genomic variants features, including effects on transcription/translation, genomic context, annotation resources, in silico pathogenicity predictions, and population allele frequency. The training set, composed of known benign or pathogenic variants, serves as a reference for these algorithms to classify new and unseen variants. Our analysis reveals a limited concordance between the prediction algorithms. Despite comparable true/false positives/negatives, discrepancies persist in variant classification. Certain algorithms exhibit a propensity to over or under-call deleterious mutations. Some demonstrate a tendency to classify random variants in non-cancer genes as deleterious. Challenges include the absence of consensus on informative features, diverse training datasets, and restriction to well annotated proteins/transcripts. Balancing the sensitivity and false positives in detecting cancer drivers is crucial. Integrating individual prediction scores with ML algorithms enhances tool performance but comes with risk of error propagation, and limited accuracy. The study emphasises the need for context-specific variant classification tools, as many variants' impacts are cancer-type specific, and some may drive disease synergistically. Existing tools, designed for a "one variant - one score approach," struggle to capture complex associations, especially those dependent on changes in the tumour microenvironment. Highlighting areas for improvement, the study addresses the "black box" problem in decision processes. While limited interpretability might not hinder practical applications, tools should evolve to assess more complex associations guided by biology. Formal consensus, reference training datasets, and standards are deemed essential for developing next-generation tools. The envisioned context-dependent tools aim to streamline feature complexity, thereby mitigating the black box problem and advancing the accuracy and interpretability of cancer variant annotation. Citation Format: Madhumita, Zbyslaw Sondka, Jon Teague. Evaluating the utility of in silico variant annotation tools for cancer driver detection [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 4884.

Abstract 6680: High-fiber diet induces favorable gut microbiota, reduces immunosuppression, and inhibits lung cancer development

Abstract Emerging evidence suggests that resident microbial communities act as a central regulator of host homeostasis. The dynamic structure, composition, and function of gut microbiota can be influenced by numerous factors, among which nutritional profile act as most crucial determinant. Microbial consortia inhabiting the gut have been implicated in the etiology and pathogenesis of various cancers and are known to influence cancer immunosurveillance and response to immunotherapy. Despite these known associations, the underlying mechanisms by which dietary interventions such as those rich in fiber (high-fiber diet) regulate gut-immune axis in prevention of lung cancer are poorly defined. Here, we employed a human-relevant mouse model in which knockout of G-protein coupled receptor 5A (Gprc5a−/−) and exposure to tobacco lead to development of preneoplasia and lung adenocarcinoma (LUAD). Mice were randomly grouped based on dietary treatment: no fiber (control), 10% cellulose, 10% pectin-inulin following weaning (early life) or after transplant of MDA-F471 NNK-induced LUAD cells (dietary intervention). Tumor growth and volumes were most significantly reduced in mice fed a high-fiber pectin-inulin diet. Notably, anti-tumor effects of the high-fiber diet were observed in both early life and later dietary intervention settings. 16S rRNA-Seq analysis of fecal pellets revealed significant differences in gut alpha as well as beta diversity, the latter indicating clear separation of community structure between fiber-deprived versus high-fiber diet groups. Evidently, gut microbiota of pectin-inulin fed mice displayed significantly elevated relative abundance of Bacteroides sartorii, Bacteroides caecimuris, Akkermansia muciniphila and Bifidobacterium pseudolongum, taxa that have been reported to be strongly associated with favorable responses to immunotherapy. High-fiber fed mice also showed significantly reduced fractions of tumor-promoting genera such as Odoribacter and Alistipes. These effects were accompanied by widespread changes in the tumor immune microenvironment between fiber-rich and fiber-poor groups. Pectin-inulin fed mice displayed enhanced antitumor immune responses evident by remarkably increased fractions of B lineage cells, cDC1s and M1-likemacrophages and, conversely, reduced levels of activated cDC2s, M2 macrophages and polymorphonuclear myeloid-derived suppressor cells. Our findings inform on novel mechanisms by which dietary fiber robustly impacts gut consortia and immune milieu in the inhibition of LUAD. Our findings also suggest that the incorporation of dietary fiber in cancer treatment (e.g., immunotherapy) could improve outcomes for patients with LUAD. Citation Format: Manvi Sharma, Yuejiang Liu, Hannah Moreno, Inti C. Reischle, Mohamed A. Jamal, Tieling Zhou, Zahraa Rahal, Chidera O. Chukwuocha, Ke Liang, Robert R. Jenq, Matthew C. Ross, Kristi L. Hoffman, Humam Kadara. High-fiber diet induces favorable gut microbiota, reduces immunosuppression, and inhibits lung cancer development [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 6680.

Abstract 187: Macrophage instead of leukemia cell expressed PD-L1 drives T cell exhaustion and immune evasion of BCRABL1 chronic myeloid leukemia

Abstract BCR-ABL1 fusion gene is the hall marker of chronic myeloid leukemia (CML). Currently, the immune microenvironment of BCRABL1 CML remains largely unexplored. Our recent investigation in a transgenic mouse model of BCRABL1 CML revealed a continuous increase in CD11b+Ly6Cint PMN-MDSC with the progression of the disease. Concurrently, there was an increase in CD11b+Ly6Chigh M-MDSC and F4/80+ macrophages. However, The levels of CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD49b+ NK cells, which are essential for tumor-killing significantly decreased. This immune phenotype was consistently observed in both primary leukemia induction and following serial transplantation of cells from primary leukemic mice. Intriguingly, only recipient mice subjected to sublethal irradiation developed the disease, while mice with intact immune system remained disease-free, which underscores the vital role of the immune system in the development of CML. The predominant presence of CD11b+Ly6Cint PMN-MDSC in the peripheral blood, bone marrow and spleen implies these cells may be at least partially consisted of CML leukemia cells. To examine the hypothesis, qRT-PCR and immunofluorescence (IF) staining were conducted. In both assays, Ly6G+ cells from BCRABL1 mice coexpressed the BCRABL1 oncogene. Furthermore, when co-cultured with CD4 T cells, the division of T cells was significantly inhibited, implying immune suppression by the Ly6G+ PMN-MDSCs and/or CML leukemia cells. In addition, the immuosuppressive pathway genes including Arg1, Hif1a, Enptd1, TGFβ were significantly upregulated. To investigate the possible role of the PD1/PDL1 axis in the BCRABL1 model, flow cytometry analysis was conducted. Unexpectedly, PDL1 was barely expressed in CML leukemia cells, whereas the expression of PDL1 in M-MDSCs and macrophages significantly increased, accompanied by remarkable upregulation of PD1 expression on CD4 T cells and CD8 T cells during leukemia progression. In other words, PDL1 expression on macrophages, rather than leukemia cells, was found to induce T cell exhaustion. For translational purpose, mice transplanted with the primary CML leukemia cells were treated with chemotherapy drugs ponatinib alone or in combination with the anti-PD1 antibody. Compared with the chemotherapy alone group, the mice in the combinational therapy group exhibited rapid remission and better disease control. Most importantly, the disease relapse occurred at a slower rate after treatment discontinuation, suggesting the value of combinational therapy in achieving treatment free remission. In summary, our study not only revealed the dynamic changes of immune microenvironment in the BCRABL1 CML mouse model, but also revealed the different mechanisms underlying immune evasion of BCR-ABL1 CML leukemia cells, which sheds light on a combinational treatment for achieving the treatment free remission of CML. Citation Format: Xiaocui Lu, Hui Fang, Xuexiu Fang, Atsuko Matsunaga, Kebin Liu, Jorge Cortes, John K. Cowell, Tianxiang Hu. Macrophage instead of leukemia cell expressed PD-L1 drives T cell exhaustion and immune evasion of BCRABL1 chronic myeloid leukemia [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 187.

Abstract 6400: Tislelizumab combined with XELOX as a neoadjuvant therapy for locally advanced gastric and gastroesophageal junction adenocarcinomas: A prospective, phase II trial

Abstract Background: Combing anti-PD-(L)1 antibody with chemotherapy in neoadjuvant therapy of gastric/gastroesophageal junction (G/GEJ) adenocarcinomas is a promising strategy, but clinical data are remain insufficient. Whether immunotherapy biomarkers in previous studies (TMB, MSI, PD-L1, etc.) have equivalent predictive value for G/GEJ remains to be verified. In this phase II clinical trial of tislelizumab in combination with chemotherapy, potential therapy-related biomarkers in tumor samples, and changes in the tumor microenvironment during treatment were analyzed, and a predictive model for efficacy was developed. Methods: This was a prospective, single-center, single-arm, phase II trial. Patients with G/GEJ adenocarcinomas (stage cT3-4aN+M0) were treated with PD-1 inhibitor tislelizumab in combination with oxaliplatin plus capecitabine (XELOX) as neoadjuvant treatment for 2 or 3 cycles. The primary endpoint was the major pathological response (MPR). The secondary endpoints included pathological complete response (pCR) rate, R0 resection rate, and safety. Tumor samples underwent RNA-sequencing (Amoy Diagnostics Co Ltd, Xiamen, China), to identify potential biomarkers, and a predictive model was developed to explore factors influencing tumor response and survival. Results: 28 patients were enrolled; mean age was 59.5 years (SD = 8.2), 19 (67.9%) patients were male; 14 (50.0%) patients with primary tumor location in the stomach, and 14 (50.0%) patients with primary tumor location in the GEJ. All patients were EB virus negative. Except one patient with PMS2 loss, 27 patients were pMMR. Nine (32.1%) patients achieved MPR after treatment, with four (14.3%) of them achieved pCR. The R0 resection rate was 96.4%, with 14 (50.0%) patients achieved objective response. No serious adverse events were reported. The most common grade 2 TRAEs during neoadjuvant treatment were anemia (5 of 28, 17.86%) and nausea (3 of 28, 10.71%). Analysis of differentially expressed genes revealed enrichment of the antigen processing and presentation pathway in the MPR group, while the TGF-β signaling pathway was enriched in the non-MPR group. Notably, the differential immune microenvironment analysis also uncovered significant differences in the TGF-β signature between the MPR and non-MPR groups. Additionally, a therapeutic response prediction model (HT score) was developed with an AUC of 0.98 (0.932-1) in the training set, and its performance was validated in publicly available datasets. Conclusion: Tislelizumab combined with XELOX holds promise as a neoadjuvant therapy for resectable G/GEJ adenocarcinomas, especially for EBV- and pMMR population, offering a well-tolerated safety profile. Moreover, the HT score has the potential to predict treatment efficacy in this regimen. Trial registration numbers: NCT05507658 and NCT05508399. Citation Format: Haikun Zhou, Tao Han, Lei Tuo, Qingchuan Yang, Ruiqi Gao, Pengfei Yu, Jiangpeng Wei, Changbin Zhu, Jin Wang, Peng Cheng, Xiang Kang, Sihui Pi, Hui Liu, Xiaoxi Pan, Xiaohua Li, Gang Ji. Tislelizumab combined with XELOX as a neoadjuvant therapy for locally advanced gastric and gastroesophageal junction adenocarcinomas: A prospective, phase II trial [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 6400.

Abstract 2593: Spatial multi-omics profiling of tumor cell phenotypes in primary cutaneous melanoma at single-cell resolution

Abstract Primary cutaneous melanomas develop in stages from melanoma precursor cells that may progress to melanoma in situ (MIS) and eventually to invasive disease. Molecular changes, such as tumor cell state switches from melanocytic to undifferentiated and neural-crest-like phenotypes, have been associated with the development of melanoma metastases and therapeutic resistance. However, it is unknown whether cell states or gene expression changes enriched in advanced tumors exist at the premalignant stage and if they can elicit tumor-infiltrating lymphocytes (TILs). To address this gap, we analyzed a cohort of clinical samples from 43 patients with primary cutaneous melanoma using whole-slide cyclic multiplex immunofluorescence imaging (CyCIF), morphology-guided micro-regional RNA sequencing, and high-resolution 3D imaging. Each specimen contained multiple histologically-distinct regions representing a progression axis from normal to precursor to melanoma in situ to invasion. Subsequently, we examined the molecular profiles of both atypical melanocytes and tumor cells, and the gene expression and tumor microenvironmental (TME) changes that occurred during these progression stages. We identified major intra- and inter-specimen heterogeneity in the tumor intrinsic and extrinsic features across progression stages. Multiple tumor samples contained regions where publicly available gene signatures linked to both melanocytic and undifferentiated cell phenotypes were enriched. CyCIF analysis confirmed that various tumor cell phenotypes can coexist in close proximity to each other in early-stage untreated melanomas, and that the majority of undifferentiated tumor cells were located within regions of dermal invasion. Moreover, the cellular neighborhoods around undifferentiated tumor cells had a higher proportion of CD8+ T cells and cells of myeloid lineage compared to the neighborhoods around melanocytic tumor cell phenotypes. When assessing the localization and abundance of various tumor cell phenotypes, the levels of spatial and regional heterogeneity increased proportionally along normal to premalignant to malignant progression. Furthermore, the regional entropy of tumor cell phenotypes decreased along tumor depth in the majority (63%) of the samples, and even the melanoma in situ regions showed varying levels of spatial heterogeneity. Our in-depth multimodal molecular analyses highlight the presence of spatial heterogeneity among tumor cell phenotypes as well as the organization of early-stage primary melanomas, and are suggestive of tumor cell adaptation to the local environment. Citation Format: Tuulia Vallius, Edward Novikov, Ajit Nirmal Johnson, Shi Yingxiao, Roxanne Pelletier, Shishir Pant, Zoltan Maliga, Alyce Chen, Clarence Yapp, Sabrina Chan, Christine Lian, George F. Murphy, Sandro Santagata, Peter Sorger. Spatial multi-omics profiling of tumor cell phenotypes in primary cutaneous melanoma at single-cell resolution [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 2593.

Abstract 6848: Analysis of the bone microenvironment cells by single cell transcriptomics reveals altered features in multiple myeloma patients compared to pre-malignant monoclonal gammopathies

Abstract Multiple myeloma (MM) is a malignant plasma cell dyscrasia that can be preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). How the bone microenvironment (BME) changes and contributes to tumoral progression in MM remains unresolved. Published data on BME are mainly produced in vitro and few data are on MGUS/SMM patients. Up to date, no single-cell RNA sequencing database of the BME cells has been described in MM. The aim of this project was to characterize for the first time at single cell level, the BME in patients with newly diagnosed MM (MMD) and MGUS/SMM to identify alterations involved in tumoral progression. From 15 bone biopsies of MMD, MGUS and SMM patients, we depleted CD235a+, CD45+, CD31+, and CD138+ cells to enrich the rare BME non-hemopoietic cells. The CD45-CD31-CD235a-CD138- cells were analyzed by scRNAseq. Data were generated on Chromium 10X Genomics. Cellranger and Seurat pipeline in R software has been used. Cell identities were assigned by manual curation. The trajectory analysis has been made by Monocle3. Decouple R and FindMarkers were used to compare pathways, and differential gene expression (DGE) of the samples throughout the different conditions. A total of 35723 cells were profiled. We identified 13 BME cell type clusters: 7 mesenchymal stromal cells (MSCs) clusters (proliferating MSC, MSC DDK-OPG-, MSC DKK+, MSC RUNX2-, MSC OPG+, MSC Osteogenic, Perivascular MSC) and 6 osteoblasts (OBs) clusters (OB precursor, OB precursor LEPR+, OB BGLAP+, OB SPP1+, OB NFkB+, OB late). Psdeudotime analyses highlighted a complex trajectory of differentiation from more immature cell clusters (proliferating MSC) to late OB cluster, confirming the ability of the dataset to snapshot the in vivo complexity of BME and we highlighted an alteration of the MSCs clusters trajectories starting in SMM samples till a disruption in MM. Pathway analyses, in MMD compared to MGUS/SMM, revealed an upregulation of VEGF and PI3K activity in proliferating MSC, perivascular MSC and OB clusters. TRAIL pathway activity, known to suppress MM cell proliferation, was upregulated in MSC osteogenic and OB BGLAP+ in MGUS. Finally, DGE analyses revealed the modulation of different genes, many not yet described in MM. MGUS MSCs clusters showed an upregulation of FBLN1, SERPNF1 and MMD MSCs clusters dramatically downregulated WISP2 and upregulated SRGN, involved in MM cell proliferation. Interestingly, in SMM samples, we reported an upregulation of genes involved both in the promotion of osteoblastogenesis (SCARA3, SPON2) and inhibition (GREM1), likely underling the attempt to contrast the establishment of altered BME, characteristic of MM. Our approach is able to dissect the complex organization of the BME and to highlight at single cells level the alterations of the BME in patients with MM compared to MGUS and SMM. Citation Format: Paola Storti, Tommaso Torelli, Cristina Manferdini, Denise Toscani, Alessandro Guidi, Rosanna Vescovini, Nicolas Thomas Iannozzi, Benedetta Dalla Palma, Matteo Scita, Federica Librale, Stefania Ricci, Vincenzo Raimondi, Oxana Lungu, Giannalisa Todaro, Gabriella Sammarelli, Emanuela Aleo, Gina Lisignoli, Luca Agnelli, Nicola Giuliani. Analysis of the bone microenvironment cells by single cell transcriptomics reveals altered features in multiple myeloma patients compared to pre-malignant monoclonal gammopathies [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 6848.

Abstract 160: FGFR1+ cancer associated fibroblasts (CAFs) produce an extracellular matrix (ECM) that curbs infiltration of phagocytic tumor-associated macrophages (TAMs)

Abstract About 40% of diffuse large B-cell lymphoma (DLBCL) pts do not respond to immunochemotherapy. In human and murine models, we and others have shown that the lymphoma microenvironment (LME) changes with disease progression and treatment resistance. This lymphoma cell-LME co-evolutionary process results in expansion of treatment-resistant lymphoma cells as well as reduced infiltration of T cells and reprogramming of CAFs and TAMs into immune suppressive entities. Our analysis of relapse or refractory (RR)-DLBCLs showed not only an immune depleted LME but also an enrichment of CAFs harboring activation of fibroblast growth factor receptor (FGFR1+). Since FGFR1 inhibitors (FGFR1i) are clinically available, we sought to determine its anti-neoplastic effect on DLBCL. We first confirmed that lymphoma cells, including murine and human, and primary and RR-DLBCLs, did not express FGFR1. Accordingly, no anti-tumoral effect was detected when the selective FGFR1i SSR128129E was administered in vitro to a lymphoma cell panel. However, oral administration of FGFR1i to pre-clinical lymphoma models including two RR-DLBCL patient-derived xenografts (PDX) and four syngeneic murine models representing distinct DLBCL genetic subtypes produced a significant anti-lymphoma effect in all cases. In addition to decreased lymphoma cells, we observed increase in the ECM and TAM infiltration, even in the murine models, suggesting that FGFR1i reprogrammed the CAFs. We thus conducted matrisome analysis (i.e., ECM proteomics) in a murine model (p53 mutant) and found a significant up-regulation of proteoglycans and the immune active glycoproteins decorin, lumican and byglican (q-value <0.05 vs. vehicle). To determine whether these ECM changes affect lymphoma cells, we performed 3D co-cultures of lymphoma cells embedded into de-celularized ECM obtained from FGFR1i vs. vehicle mice. We found that FGFR1i-ECM (vs. vehicle-ECM) modifies the secretome of lymphoma cells by upregulating several cytokines including IL6, IL16 and TNF-alpha. To further characterize the functional state of CAFs and TAMs, we conducted single cell RNA-seq in the two RR-DLBCL PDXs treated as before. We found that FGFR1-CAFs in the LME of FGFR1i tumors (vs. vehicle) were reprogrammed into inflammatory-like CAFs characterized by the expression of several monocyte/macrophage chemotactic factors including CCL2, CCL7, CXCL3, and CXCL12. Consequent with these findings we not only observed an increase infiltration of TAMs in the LME of FGFR1i tumors but also that they were reprogrammed in phagocytic phenotypes. In sum, our results suggest that CAF-FGFR1+ produce an immune “silent” ECM that characterizes RR-DLBCL while FGFR1i reverses this ECM phenotype ultimately resulting in the infiltration of antitumoral TAMs, delineating a novel ECM-directed therapeutic approach for this disease. Citation Format: Nicolás Di Siervi, Maria Victoria Revuelta, Nahuel Zamponi, Giorgio Inghirami, Leandro Cerchietti. FGFR1+ cancer associated fibroblasts (CAFs) produce an extracellular matrix (ECM) that curbs infiltration of phagocytic tumor-associated macrophages (TAMs) [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 160.

Microenvironmental changes in familial adenomatous polyposis during colorectal cancer carcinogenesis

Familial adenomatous polyposis (FAP) is a heritable disease that increases the risk of colorectal cancer (CRC) development because of heterozygous mutations in APC. Little is known about the microenvironment of FAP. Here, single-cell RNA sequencing was performed on matched normal tissues, adenomas, and carcinomas from four patients with FAP. We analyzed the transcriptomes of 56,225 unsorted single cells, revealing the heterogeneity of each cell type, and compared gene expression among tissues. Then we compared the gene expression with that of sporadic CRC. Furthermore, we analyzed specimens of 26 FAP patients and 40 sporadic CRC patients by immunohistochemistry. Immunosuppressiveness of myeloid cells, fibroblasts, and regulatory T cells was upregulated even in the early stages of carcinogenesis. CD8+ T cells became exhausted only in carcinoma, although the cytotoxicity of CD8+ T cells was gradually increased according to the carcinogenic step. When compared with those in the sporadic CRC microenvironment, the composition and function of each cell type in the FAP-derived CRC microenvironment had differences. Our findings indicate that an immunosuppressive microenvironment is constructed from a precancerous stage in FAP.

Immune checkpoint inhibitor monotherapy is sufficient to promote microenvironmental normalization via the type Iinterferon pathway in PD-L1-expressing head and neck cancer.

Immune checkpoint blockers (ICBs) targeting programmed cell death protein 1 (PD-1) have been proven to be an effective first-line therapy against programmed cell death 1 ligand 1 (PD-L1; also known as CD274 molecule)-expressing head and neck squamous cell carcinoma (HNSCC) in recent KEYNOTE-048 trial. However, associated changes in the tumor microenvironment (TME) and underlying mechanisms remain elusive. Oral tumors in C57/BL6 mice were induced by administering 7,12-dimethylbenzanthracene into the buccal mucosa. Single-cell suspension was isolated from tumor tissue; proliferating cells were injected subcutaneously into the left flank of mice to establish Ajou oral cancer (AOC) cell lines. Subsequently, a syngeneic PD-L1-expressing HNSCC model was developed by injecting AOC cells into the buccal or tongue area. The model recapitulated human HNSCC molecular features and showed reliable in vivo tumorigenicity with significant PD-L1 expression. ICB monotherapy induced global changes in the TME, including vascular normalization. Furthermore, the antitumor effect of ICB monotherapy was superior to those of other therapeutic agents, including cisplatin and inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2). The ICB-induced antitumorigenicity and TME normalization were alleviated by blocking the type I interferon pathway. In summary, ICB monotherapy is sufficient to induce TME normalization in the syngeneic model; the type I interferon pathway is indispensable in realizing the effects of ICBs. Furthermore, these results explain the underlying mechanism of the efficacy of ICB monotherapy against PD-L1-expressing HNSCC in the KEYNOTE-048 trial.

NK cell receptor profiling of endometrial and decidual NK cells reveals pregnancy-induced adaptations.

Natural killer (NK) cells, with a unique NK cell receptor phenotype, are abundantly present in the non-pregnant (endometrium) and pregnant (decidua) humanuterine mucosa. It is hypothesized that NK cells in the endometrium areprecursors for decidual NK cells present during pregnancy. Microenvironmental changes can alter the phenotype of NK cells, but it is unclear whether decidual NK cell precursors in the endometrium alter their NK cell receptor repertoire under the influence of pregnancy. To examine whether decidual NK cell precursors reveal phenotypic modifications upon pregnancy, we immunophenotyped the NK cell receptor repertoire of both endometrial and early-pregnancy decidual NK cells using flow cytometry. We showed that NK cells in pre-pregnancy endometrium have a different phenotypic composition compared to NK cells in early-pregnancy decidua. The frequency of killer-immunoglobulin-like receptor (KIR expressing NK cells, especially KIR2DS1, KIR2DL2L3S2, and KIR2DL2S2 was significantly lower in decidua, while the frequency of NK cells expressing activating receptors NKG2D, NKp30, NKp46, and CD244 was significantly higher compared to endometrium. Furthermore, co-expression patterns showed a lower frequency of NK cells co-expressing KIR3DL1S1 and KIR2DL2L3S2 in decidua. Our results provide new insights into the adaptations in NK cell receptor repertoire composition that NK cells in the uterine mucosa undergo upon pregnancy.

Unraveling the Connection: Pancreatic Cancer Cells and Schwann Cells.

Pancreatic ductal adenocarcinoma is one of the most lethal solid malignancies, characterized by its aggressiveness and metastatic potential, with a 5-year survival rate of only 13%. Progress in the management of metastatic disease has been modest. A robust connection between nervous system and tumor progression exists, with prominent neural alterations having been observed during pancreatic cancer's progression, including neural hypertrophy, neural density, and neural remodeling. The pancreatic tumor microenvironment includes s set of cells and structures that constantly dialogue with cancer cells, influencing its growth and behavior. The microglia is key cellular components of the tumor microenvironment, and Schwann cells are the principal glial cells in the peripheral neural system. Schwann cells can regulate changes in the tumor microenvironment and immune responses by secreting a variety of factors and can support a tumor's invasion of nerves and distant metastasis, with further pain exacerbation. Schwann cells secrete various pain-related molecules, such as the neural growth factor, to mediate the activation of primary sensory neurons, leading to pain induction. The binding of the neural growth factor to tropomyosin receptor kinase A is an important signaling mechanism for pain perception in humans. Consequently, directing efforts towards targeting neural invasion may provide an alternative strategy to improve the prognosis of and alleviate pain in patients with pancreatic cancer.