Cytotoxic T-cell Infiltration Research Articles

Monocyte/Macrophage-Mediated Transport of Dual-Drug ZIF Nanoplatforms Synergized with Programmed Cell Death Protein-1 Inhibitor Against Microsatellite-Stable Colorectal Cancer.

Microsatellite-stable colorectal cancer (MSS-CRC) exhibits resistance to programmed cell death protein-1 (PD-1) therapy. Improving the infiltration and tumor recognition of cytotoxic T-lymphocytes (CTLs) is a promising strategy, but it encounters huge challenges from drug delivery and mechanisms aspects. Here, a zeolitic imidazolate framework (ZIF) coated with apoptotic body membranes derived from MSS-CRC cells is engineered for the co-delivery of ginsenoside Rg1 (Rg1) and atractylenolide-I (Att) to MSS-CRC, named as Ab@Rg1/Att-ZIF. This system is selectively engulfed by Ly-6C+ monocytes during blood circulation and utilizes a "hitchhiking" mechanism to migrate toward the core of MSS-CRC. Ab@Rg1/Att-ZIF undergoes rapid disassembly in the tumor, released Rg1 promotes the processing and transportation of tumor antigens in dendritic cells (DCs), enhancing their maturation. Meanwhile, Att enhances the activity of the 26S proteasome complex in tumor cells, leading to increased expression of major histocompatibility complex class-I (MHC-I). These coordinated actions enhance the infiltration and recognition of CTLs in the center of MSS-CRC, significantly improving the tumor inhibition of PD-1 treatment from ≈5% to ≈69%. This innovative design, involving inflammation-guided precise drug co-delivery and a rational combination, achieves synergistic engineering of the tumor microenvironment, providing a novel strategy for successful PD-1 treatment of MSS-CRC.

Fecal microbiota transplantation improves anti-PD-1 inhibitor efficacy in unresectable or metastatic solid cancers refractory to anti-PD-1 inhibitor

The gut microbiome significantly influences immune responses and the efficacy of immune checkpoint inhibitors. We conducted a clinical trial (NCT04264975) combining an anti-programmed death-1 (PD-1) inhibitor with fecal microbiota transplantation (FMT) from anti-PD-1 responder in 13 patients with anti-PD-1-refractory advanced solid cancers. FMT induced sustained microbiota changes and clinical benefits in 6 of 13 patients, with 1 partial response and 5 stable diseases, achieving an objective response rate of 7.7% and a disease control rate of 46.2%. The clinical response correlates with increased cytotoxic Tcells and immune cytokines in blood and tumors. We isolated Prevotella merdae Immunoactis from a responder to FMT, which stimulates Tcell activity and suppresses tumor growth in mice by enhancing cytotoxic Tcell infiltration. Additionally, we found Lactobacillus salivarius and Bacteroides plebeius may inhibit anti-tumor immunity. Our findings suggest that FMT with beneficial microbiota can overcome resistance to anti-PD-1 inhibitors in advanced solid cancers, especially gastrointestinal cancers.

Light-Activated In Situ Vaccine with Enhanced Cytotoxic T Lymphocyte Infiltration and Function for Potent Cancer Immunotherapy.

In situ cancer vaccination is an attractive strategy that stimulates protective antitumor immunity. Cytotoxic T lymphocytes (CTLs) are major mediators of the adaptive immune defenses, with critical roles in antitumor immune response and establishing immune memory, and are consequently extremely important for in situ vaccines to generate systemic and lasting antitumor efficacy. However, the dense extracellular matrix and hypoxia in solid tumors severely impede the infiltration and function of CTLs, ultimately compromising the efficacy of in situ cancer vaccines. To address this issue, a robust in situ cancer vaccine, Au@MnO2 nanoparticles (AMOPs), based on a gold nanoparticle core coated with a manganese dioxide shell is developed. The AMOPs modulated the unfavorable tumor microenvironment (TME) to restore CTLs infiltration and function and efficiently induced immunogenic cell death. The Mn2+-mediated stimulator of the interferon genes pathway can be activated to further augment the therapeutic efficacy of the AMOPs. Thus, the AMOPs vaccine successfully elicited long-lasting antitumor immunity to considerably inhibit primary, recurrent, and metastatic tumors. This study not only highlights the importance of revitalizing CTLs efficacy against solid tumors but also makes progress toward overcoming TME barriers for sustained antitumor immunity.

In-situ-formed immunotherapeutic and hemostatic dual drug-loaded nanohydrogel for preventing postoperative recurrence of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent malignancy characterized by an exceedingly high recurrence rate post-surgery, significantly impairing the prognosis of HCC patients. However, a standard in-care strategy for postoperative therapy is still lacking. Although encouraging results have been obtained in a newly published clinical trial for postoperative therapy by targeting the vascular endothelial growth factor (VEGF) and programmed death ligand 1 (anti-PD-L1), its efficacy remains constrained. Combining a hemostatic hydrogel with a nanoparticle-based drug delivery system presents an opportunity to optimize the antitumor effect. Herein, we developed a nanoplatform, termed HMSN@Sor/aP@Gel, comprising a hemostatic fibrin hydrogel and functionalized hollow mesoporous silica nanoparticles (HMSNs) loaded with sorafenib and anti-PD-L1 for locally administered targeted-immunotherapy to prevent the postoperative recurrence and metastasis of HCC. The antitumor mechanism is grounded in dual inhibition of Ras/Raf/MEK/ERK (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathways, synergistically complemented by PD-L1 blockade. HMSN@Sor/aP@Gel facilitates dendritic cell maturation, enhances cytotoxic T-lymphocyte infiltration, promotes the polarization of tumor-associated macrophages to M1 phenotype, induces tumor immunogenic cell death, reverses immunosuppression, and establishes immune memory to counter postoperative recurrence. Animal studies corroborate that HMSN@Sor/aP@Gel-mediated targeted immunotherapy significantly impedes primary and metastatic tumor growth and establishes immune memory to prevent recurrence post-surgery. This investigation presents a promising strategy for postoperative therapy with considerable potential for clinical translation.

A dual-enzyme-like photosensitive nanozyme for remodeling the tumor immunosuppressive microenvironment to enhance immunotherapy

In “immune-cold” tumors, the upregulation of immunosuppressive cells and insufficient infiltration of lymphocytes contribute to the resistance against immune therapy. Herein, we have developed a dual-enzyme-like photosensitive nanozyme (PBAF) to remodel the tumor immunosuppressive microenvironment (TIME) and induce the tumor infiltration of cytotoxic T lymphocytes (CTLs). Specifically, PBAF exhibits peroxidase (POD)-like activity and glutathione oxidase (GSHOx)-like activity and can be stimulated by 750 nm laser, promoting oxidative stress at the tumor site. Consequently, this process further leads to the reconstruction of TIME in animal experiments, inducing tumor-associated macrophages (TAMs) toward the immunostimulatory M1 phenotype, eliminating myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Simultaneously, PBAF also promotes dendritic cells (DCs) maturation to enhance CTLs infiltration into the tumor. The remodeled TIME and enhanced immune responses by PBAF demonstrate significant post-administration inhibition of recurrence and metastasis in the treatment of malignant tumors.

Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1.

Increased levels of plasminogen activator inhibitor-1 (PAI-1) in tumors have been found to correlate with poor clinical outcomes in patients with cancer. Although abundant data support the involvement of PAI-1 in cancer progression, whether PAI-1 contributes to tumor immune surveillance remains unclear. The purposes of this study are to determine whether PAI-1 regulates the expression of immune checkpoint molecules to suppresses the immune response to cancer and demonstrate the potential of PAI-1 inhibition for cancer therapy. The effects of PAI-1 on the expression of the immune checkpoint molecule programmed cell death ligand 1 (PD-L1) were investigated in several human and murine tumor cell lines. In addition, we generated tumor-bearing mice and evaluated the effects of a PAI-1 inhibitor on tumor progression or on the tumor infiltration of cells involved in tumor immunity either alone or in combination with immune checkpoint inhibitors. PAI-1 induces PD-L1 expression through the JAK/STAT signaling pathway in several types of tumor cells and surrounding cells. Blockade of PAI-1 impedes PD-L1 induction in tumor cells, significantly reducing the abundance of immunosuppressive cells at the tumor site and increasing cytotoxic T-cell infiltration, ultimately leading to tumor regression. The anti-tumor effect elicited by the PAI-1 inhibitor is abolished in immunodeficient mice, suggesting that PAI-1 blockade induces tumor regression by stimulating the immune system. Moreover, combining a PAI-1 inhibitor with an immune checkpoint inhibitor significantly increases tumor regression. PAI-1 protects tumors from immune surveillance by increasing PD-L1 expression; hence, therapeutic PAI-1 blockade may prove valuable in treating malignant tumors.

Abstract PO1-18-03: Chemokine-modulating regimen (rintatolimod, IFN-α2b, celecoxib): New strategy to drive CD8+T-cells into triple negative breast cancer

Abstract BACKGROUND: Pathologic complete response (pCR) is critical for positive long-term outcomes after neoadjuvant chemotherapy (NAC) in triple negative breast cancer (TNBC) but is achieved only in 40-50% of patients. Combination of NAC with pembrolizumab, the new standard of care in TNBC, increases the pCR rate to 65% but is associated with significant and often permanent immune-related toxicities. Higher levels of CD8+ cytotoxic T-lymphocytes (CTLs) and low levels of regulatory T-cells (Treg) and myeloid derived suppressor cells (MDSC) in the tumor microenvironment (TME) predict improved relapse-free survival (RFS), overall survival (OS) and pCR, a surrogate marker for RFS. Locally produced chemokines CCL5, CXCL9, CXCL10 and CXCL11 are critical for CTL infiltration, while CCL22 is responsible for Treg attraction, with high CXCL9 being associated with a 3-fold higher rate of pCR after NAC. Our preclinical data show that Chemokine-modulatory (CKM) regimen, combining rintatolimod (TLR3 agonist), interferon (IFN)α2b and celecoxib (COX-2 inhibitor), eliminates the suppressive aspects of paclitaxel-induced inflammation and induces CTL-attractants but decreases MDSC- and Treg-attractants in the TME. We hypothesized that the combination of CKM with paclitaxel-based NAC will promote selective CTL infiltration into TNBC, and along with doxorubicin/cyclophosphamide (AC), will result in higher rate of pCR, translating into improved RFS and OS. METHODS: In this phase I study NCT04081389, 9 pts with stage I-III TNBC and median age of 47 (37-55) years received 3 weeks of paclitaxel with CKM, followed by 9 weeks of paclitaxel alone, followed by standard dose-dense AC and surgery. Paclitaxel was given weekly IV at 80 mg/m2 for 12 weeks, rintatolimod 200 mg IV, IFN-α2b and celecoxib 200 mg oral twice daily on days 1-3 of each of the initial 3 weeks. IFN-α2b was administered in an accelerated titration design at doses 0 or 5 million units (MU)/m2 [Dose Level (DL) 1 and 2, respectively] in the first 2 pts (no intra-patient dose escalation), then 10 MU/m2 [DL3] in 4 pts and then 20 MU/m2 [DL4] in 3 pts. Day 1 pre- and post-treatment blood samples were drawn. Pre- and post-treatment (at 3 weeks) biopsies were obtained from 5 pts at DL3 and DL4. Dose-limiting toxicity (DLT) was defined as grade 3 or higher within the first 3 weeks of treatment. Primary endpoint was safety and tolerability, to determine the recommended phase II dose (RP2D) of CKM for extended efficacy study. Secondary endpoints included efficacy (pCR), along with RFS and OS. Biomarkers were analyzed in exploratory studies. RESULTS: Treatment was well-tolerated with mostly grade 1 or 2 treatment-related adverse events (TRAEs) and no DLTs. Grade 3 TRAE were neutropenia (3/9), attributed to CKM (1/9) or paclitaxel (3/9), pneumonia (1/9) and anemia (1/9) attributed to AC. Paclitaxel- or AC-related toxicities were not higher than expected. There were no delayed or immune-related toxicities beyond treatment. 5/9 (56%) pts attained pCR, and 1 additional pt had ypTmic at the time of surgery. There was consistent (p=0.07) selective increase in CD8α (CTL marker) in post-treatment tumor biopsies with concomitant decrease in CD8α in the blood (p=0.04) measured by RT-PCR. RNA sequencing on the blood samples (7 pre- and post-treatment) showed that higher M2 macrophages and naïve B cells in the pre-treatment samples were associated with higher probability of non pCR (adjusted p=0.02 and 0.05, respectively). CKM treatment decreased CD8α and CD8β post-treatment in the blood samples. Higher CD8+ T cells and NK cells in the post-treatment blood samples, were associated with higher likelihood of pCR, although this trend did not reach statistical significance. CONCLUSIONS: We have observed preliminary indications of safety and good tolerability of the combination of CKM regimen with paclitaxel, with promising pCR + ypTmic of 66%. RNA sequencing indicates that CKM may drive CD8+ T cells from the blood into the tumor. Citation Format: Shipra Gandhi, Cayla Jones, Mateusz Opyrchal, Ronald Slomba, Kristopher Attwood, Jianmin Wang, Eduardo Cortes Gomez, Tracey O'Connor, Ellis Levine, Pawel Kalinski. Chemokine-modulating regimen (rintatolimod, IFN-α2b, celecoxib): New strategy to drive CD8+T-cells into triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-03.

Abstract PS07-07: The RXR agonist, IRX4204, delays the formation of Brca1 mutant mammary tumors via modulation of the anti-tumor immune response

Abstract Background: Women with germline BRCA1 mutations are at an increased risk for developing breast cancer in their lifetime, often at a young age with more aggressive tumors. At present, prophylactic bilateral mastectomy is the most effective strategy for reducing breast cancer risk. However, this invasive procedure is irreversible and associated with potential complications. We and others have found that PARP inhibitors can delay Brca1-mutant tumor formation in mice and could be beneficial for the prevention of breast cancer. However, currently available PARP inhibitors are associated with modest toxicities that may not be acceptable to women without cancer. Thus, there remains an urgent need for the development of safe and effective therapies for the prevention of breast cancer. Here, we present data demonstrating the activity of IRX4204, a minimally toxic and highly specific agonist of the nuclear retinoid X receptor (RXR), to delay the formation of mammary tumors in a Brca1-deficient mouse model. This inhibitory effect on tumor growth is due, in part, to the role of IRX4204 in stimulating the anti-tumor immune response. Methods: We used the established MMTV-Cre, conditional Brca1 gene knockout, p53 heterozygous loss mouse model (BRCA1co/co; MMTV-Cre+/+; p53+/-) and selected for mutant female pups using PCR genotyping. At 16 weeks of age, all mice were separated into 4 treatment groups (n=10 per group): (1) sesame oil control; (2) the novel RXR agonist, IRX-4204 (10mg/kg); (3) high-dose IRX4204 (20 mg/kg); and (4) the RXR agonist, 9-cis-UAB-30 (5 mg/kg). All treatments were given by oral gavage five days per week, and mice were observed daily for tumor formation and toxicity. At the study endpoint, tumors and normal mammary glands were collected for additional analyses. Immunohistochemical staining was used to quantify CD8a, Ki-67 and cleaved caspase 3 expression in Brca1-deficient tumors. Oil Red O staining was used to measure changes in lipid accumulation in Brca1-deficient cell lines treated with IRX4204. qPCR was used to quantify the changes in gene expression of lipid metabolism-associated genes upon treatment with IRX4204 in vitro. Results: Vehicle-treated Brca1-deficient mice had a median time-to-tumor formation (TTF) of 211 days, with 100% developing tumors by 330 days. Mice treated with UAB 5 mg/kg had an improved median TTF of 261 days, whereas mice treated with IRX4204 10mg/kg or 20mg/kg had a median TTF of 347 and 304 days, respectively (p < 0.01). In addition, 60% of mice treated with IRX4204 10 mg/kg remained tumor-free at 330 days. IRX4204-treated tumors showed an increased infiltration of CD8-positive T-cells over vehicle-treated tumors (p < 0.05). Treatment of Brca1-deficient cell lines with IRX4204 in vitro resulted in a significant increase in lipid accumulation accompanied by a 2-fold increase of Srebf1 expression (a key transcription factor that regulates lipid homeostasis) within 24 hours of treatment (p < 0.05). Conclusion: These data demonstrate a novel use of the RXR agonist, IRX4204, to delay the formation of Brca1-deficient mammary tumors. We have found that IRX4204 treatment modulates the tumor immune response through increased infiltration of cytotoxic CD8-positive T-cells in Brca1-deficient mammary tumors in vivo. We have also determined that IRX4204 modulates lipid metabolism in breast cancer cell lines in vitro. It is known that lipid-derived antigens can stimulate T-cell activity. Our findings suggest that RXR agonists may alter lipid antigen production to activate an anti-tumor response. Additional immune and lipidomic studies are on-going. This work was supported by NCI-PREVENT grant (to PB and AM HHSN26100008) and CFP Foundation (Odyssey Fellowship to CM). Citation Format: Cassandra Moyer, Darian Coleman, Jamal Hill, Lana A. Vornik, Michelle Savage, Martin Sanders, Sei Shizuko, Altaf Mohammed, Powel Brown, Abhijit Mazumdar. The RXR agonist, IRX4204, delays the formation of Brca1 mutant mammary tumors via modulation of the anti-tumor immune response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-07.

NIR-responsive copper nanoliposome composites for cascaded ferrotherapy via ferroptosis actived ICD and IFN-γ released

Metallic biomaterials activate tumor ferroptosis by increasing oxidative stress, but their efficacy is severely limited in tumor microenvironment. Although interferon gamma (IFN-γ) can promote tumor ferroptosis sensitivity by inhibiting the antioxidant system and promoting lipid accumulation, this effect limited by the lack of IFN-γ accumulation in tumors. Herein, we report a near-infrared (NIR)-responsive HCuS nanocomposite (HCuS-PE@TSL-tlyp-1) that can stimulate immunogenic cell death (ICD)-mediated IFN-γ secretion through exogenous oxidative stress, thereby achieving cascaded ferrotherapy by mutually reinforcing ferroptosis and systemic immunity. Upon laser irradiation, the dissolution of the thermal coating, and the introduction of Cu ions and piperazine-erastin (PE) simultaneously induce oxidative stress by reactive oxygen species (ROS)/lipid peroxide (LPO) accumulation and deplete cystine-glutamate transporter (xCT)/GSH. The onset of oxidative stress-mediated ferroptosis is thus achieved, and ICD is triggered, significantly promoting cytotoxic T-cell (CTL) infiltration for IFN-γ secretion. Furthermore, IFN-γ induces immunogenic tumor ferroptosis by inhibiting xCT-antioxidant pathways and enhancing the ACSL4-fatty acid recruitment pathway, which further promotes sensitivity to ferroptosis in cells. These HCuS nanocomposites combined with aPD-L1 effectively in inhibiting tumor metastasis and recurrence. Importantly, these cascade ferrotherapy results broadens the application of HCuS biomaterials.

Abstract NG01: Germline-mediated immunoediting sculpts breast cancer subtypes and metastatic proclivity

Abstract NG01: Germline-mediated immunoediting sculpts breast cancer subtypes and metastatic proclivity

Tumor microenvironment reprogramming combined with immunogenic enhancement by nanoemulsions potentiates immunotherapy

The combination of immune checkpoint inhibitors and immunogenic cell death (ICD) inducers has become a promising strategy for the treatment of various cancers. However, its efficacy remains unmet because of the dense stroma and defective vasculatures in the tumor microenvironment (TME) that restricts the intratumoral infiltration of cytotoxic T lymphocytes (CTLs). Herein, cancer-associated fibroblasts (CAFs)-targeted nanoemulsions are tailored to combine the ICD induction and the TME reprogramming to sensitize checkpoint blockade immunotherapy. Melittin, as an ICD inducer and an antifibrotic agent, is efficiently encapsulated into the nanoemulsion accompanied by a nitric oxide donor to improve its bioavailability and tumor targeting. The nanoemulsions exhibited dual functionality by directly inducing direct cancer cell death and enhancing the tumoral immunogenicity, while also synergistically reprogramming the TME through reversing the activated CAFs, decreasing collagen deposition and restoring tumor vessels. Consequently, these nanemulsions successfully facilitated the CTLs infiltration and suppressing the recruitment of immunosuppressive cells. A combination of AE-MGNPs and anti-CTLA-4 antibody greatly elicited a striking level of antitumor T-cell response to suppress tumor growth in CAFs-rich colorectal tumor models. Our work emphasized the integration of the ICD induction with simultaneous modulation of the TME to enhance the sensitivity of patients to checkpoint blockade immunotherapy.

RNAi mediated silencing of STAT3/PD-L1 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors

Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical rodent tumor models, chemically stabilized acylated small interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types, reduced STAT3 protein levels, and increased cytotoxic Tcell infiltration. In a murine model of CPI-resistant pancreatic cancer, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced in combination with CPIs. To further exemplify the utility of RNAi for cancer immunotherapy, this technology was used to silence Cd274, the gene encoding the immune checkpoint protein programmed death-ligand 1 (PD-L1). Interestingly, silencing of Cd274 was effective in tumor models that are resistant to PD-L1 antibody therapy. These data represent the first demonstration of systemic delivery of RNAi agents to the TME and suggest applying this technology for immuno-oncology applications.

Abstract 6127: Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals

Abstract Background: Approximately 50% of high-grade serous ovarian carcinomas (HGSOC) are homologous recombination deficient (HRD). HRD is associated with increased T-cell infiltration and improved survival. This study aimed to describe the distribution of HRD features and cytotoxic T-cell infiltration by gene expression subtype among Black HGSOC cases. Methods: We included Black HGSOC cases from the African American Cancer Epidemiology Study and the North Carolina Ovarian Cancer Study. We used RNASeq data to assign gene expression subtypes (n=160), and whole exome sequencing data from matched blood and tumor formalin-fixed paraffin-embedded specimens (n=206) to characterize HRD features. We defined HRD features as (1) germline variants and somatic mutations in HRD genes, and (2) HRD-associated signatures (SBS3 and ID6) that we obtained from de novo mutational signature analysis for single-base pair substitution, insertion, and deletion. We estimated the proportion of intratumoral cytotoxic T cells using multiplex immunofluorescence (n=125). Results: The subtype distribution in our study was 39% immunoreactive, 29% mesenchymal 27% proliferative, and 4% differentiated. Germline variants and somatic mutations in any HRD gene were observed in 27% and 23% of cases, respectively, and at least one of these was observed in 42% of cases. Pathogenic germline variants and somatic mutations were observed in 7% and 3% of cases, respectively. The prevalence of germline or somatic variants by gene was 16% for BRCA1, 14% for BRCA2, 11% for ATM, 5% for RAD51, and 2% each for BRIP1, PALB2, and BARD1. We observed a COSMIC HRD signature in 43% of cases, with 25% SBS3 only, 5% ID6 only, and 13% with both signatures. The prevalence of any HRD gene variant or signature was higher in immunoreactive cases (52% and 59%, respectively) than in mesenchymal (28% and 43%) or proliferative (44% and 28%) cases. Germline HRD variants were more common in the proliferative subtype (44%) compared with immunoreactive (25%) and mesenchymal (17%). In contrast, somatic HRD mutations were more common in the immunoreactive subtype (33%), compared with proliferative (16%) and mesenchymal (15%). This was due to a higher occurrence of BRCA1 somatic mutations in the immunoreactive subtype (25%) compared with proliferative (9%) and mesenchymal (8%). A higher proportion of cytotoxic T-cells (≥1% vs <1%) was also observed in the immunoreactive subtype (44%) compared with mesenchymal (31%) and proliferative (11%). Conclusion: We observed differences in the distribution of HRD features and cytotoxic T-cells by gene expression subtype. In particular, in the immunoreactive subtype, the most common gene expression subtype in Black HGSOC cases, we observed a higher occurrence of any HRD feature, somatic BRCA1 mutations, and cytotoxic T cell infiltration than in any of the other gene expression subtypes. Citation Format: Katherine Lawson-Michod, Mollie E. Barnard, Natalie Davidson, Lindsay J. Collin, Courtney Johnson, Lucas A. Salas, Casey Greene, Jeffrey R. Marks, Lauren Peres, Joellen M. Schildkraut, Jennifer A. Doherty. Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals [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 6127.

Abstract 5182: Exploiting tumor RNA-sequencing data for prediction of immune checkpoint inhibition response

Abstract Immune checkpoint inhibitors (ICI) have become the standard of care as a first- or second-line systemic treatment for patients with various cancer types. Unfortunately, many patients do not respond to this treatment and the occurrence of immune-related adverse events varies widely. There is an urgent need for robust predictive biomarkers for ICI response to identify patients with likely clinical benefit from this costly treatment. Several biomarkers, aimed at predicting response to ICI, have been proposed, including PD-L1 expression, tumor mutation burden (TMB), infiltration of cytotoxic T-cells in the tumor, Microsatellite Instability (MSI) and expression of various immune gene signatures. However, these individual biomarkers have suboptimal performance and multiple complementary omics technologies are required to properly quantify them. Integration of these multi-omic biomarkers has proven valuable in increasing the accuracy and robustness of ICI response prediction.We have developed a computational pipeline that determines the expressed mutation burden (eTMB), MSI status, fraction of infiltrating immune cells and various immune gene expression signatures directly from the RNA-sequencing profile of the tumor. Each biomarker is quantified using a dedicated algorithm that applies machine learning (eTMB and MSI) or computational deconvolution (infiltrating immune cells) and integrates external data sources to maximize performance. Algorithm performance has been validated on large cohorts of tumor RNA-sequencing data with matching gold standard quantification of said biomarkers. As a proof of concept, we applied our pipeline to tumor RNA-sequencing data of 45 gastric cancer patients treated with pembrolizumab. Responders showed significantly higher eTMB scores and were enriched among the MSI-H patients. Fractions of CD8 T-cells and M1 Macrophages, together with interferon gamma and cytotoxic T-cell gene expression signatures, were significantly increased in responders. Notably, integrating these biomarkers into a single prediction model improved prediction of response to checkpoint inhibition therapy compared to predictions based on eTMB or MSI alone. Taken together, our approach enables the quantification of various ICI response biomarkers from a single omics layer (RNA-sequencing) and can contribute to ICI response prediction. Citation Format: Pieter Mestdagh, Pieter-Jan Van Dam, Frederick De Baene, Carolina Fierro, Elise Van Hoof, Emmanuel Rivière, Hanne Dangreau, Reindert Van Cauwenberge, Jo Vandesompele. Exploiting tumor RNA-sequencing data for prediction of immune checkpoint inhibition response [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 5182.

Abstract 4178: Non-invasive shortwave infrared imaging of cytotoxic T lymphocyte infiltration for monitoring responses to combination immunotherapy and chemotherapy

Abstract Although triple-negative breast cancer (TNBC) can be treated with anti-PD-1 checkpoint immunotherapy in combination with chemotherapy, there remains a challenge in effectively monitoring therapeutic responses. Current non-invasive clinical imaging tools to evaluate response to treatment are reliant upon measurements of tumor volume and may fail to distinguish true progression from increased immune cell infiltration. Invasive biopsy sampling and immunohistochemistry (IHC) can elucidate changes in the immune landscape of treated tumors, but these methods are not conducive to providing real-time information. This study presents shortwave infrared (SWIR) imaging as a potential tool to detect treatment-induced cytotoxic T lymphocyte (CTL) infiltration non-invasively and in real time using rare earth metal-doped nanoparticles encapsulated in human serum albumin nanocomposites (ReANCs). ReANCs were chemically conjugated with anti-CD8α antibodies as targeting ligands to facilitate binding of the nanoprobes to CTLs with high specificity in a syngeneic mouse model of breast cancer. After treating the mice with combination anti-PD-1 and doxorubicin, volumetric analysis of the mammary fat pad tumors did not show any significant impact of treatment compared to single treatment and untreated control mice. However, increased CTL infiltration in the tumors of mice that received combination treatment was detected by in vivo SWIR imaging. CTL infiltration was validated with ex vivo IHC staining, and a monotonic relationship was observed between SWIR fluorescence and CD8 positivity. IHC staining of other immune markers, including CD45, CD3, CD4, and PD-L1, showed that combination treatment may influence in the expression of these markers, presenting additional targets that could be imaged with ReANCs in the future. In conclusion, the increase in SWIR signal from CD8-targeted ReANCs in tumors treated with combination immunotherapy and chemotherapy and the relationship with IHC staining highlight the ability to use SWIR imaging for non-invasive assessment of changes in immune dynamics following treatment. Citation Format: Jay V. Shah, Jake N. Siebert, Xinyu Zhao, Shuqing He, Richard E. Riman, Mei Chee Tan, Mark C. Pierce, Edmund C. Lattime, Vidya Ganapathy, Prabhas V. Moghe. Non-invasive shortwave infrared imaging of cytotoxic T lymphocyte infiltration for monitoring responses to combination immunotherapy and chemotherapy [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 4178.

Abstract 1594: Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response

Abstract Background: While T-cell-based immunotherapy emerged as a promising tool for cancer management, clinical trials and translational studies revealed that cancer cells develop resistance by modulating tumor microenvironment and cell-intrinsic mechanisms. Pancreatic cancer (PC), consisting of dense stroma, immunosuppressive environment, and aberrant mucin expression, has a dismal survival rate and responds poorly to immunotherapies. MUC4, a member of the mucin family, has been reported to block lymphokine-activated killer cells and induce apoptosis of cytotoxic T-cells, suggesting its possible role in immune modulation. However, how the T-cell secretome influences MUC4 expression in PC and its role in modulating the T-cell response is poorly investigated. Method: The scRNA-seq data analysis and primary T-cell conditioned media (CM) were utilized to investigate the MUC4 and T-cell crosstalk. T-cell CM-treated murine PC cell line (KCT-3266) was analyzed by RNA-seq analysis followed by MUC4 silencing studies to investigate its role in evading T-cell response. The subcutaneous murine model was utilized to investigate the influence of MUC4 KO on immune cells and cytotoxic cell infiltration. The RNA isolated from tumor tissues from KrasG12D/+, Trp53R127H/+, Pdx-1-Cre (KPC), and Muc4 knockout (KPMC) murine models were used for PanCancer immune profiling. Results: The scRNA-seq analysis suggests that intratumoral T-cells positively correlate with MUC4 expression in PC patients. The activated T-cell CM consisting of predominantly IL-2, IFN-γ, and TNF-α cytokines, significantly induce the expression of MUC4 transcriptionally and translationally in both human (SW1990 and COLO357) and murine (KCT-3248 and KCT3266) PC cell lines. RNA-seq analysis from T-cell CM-treated cancer cells revealed that T-cell secretome induced the pathways related to cancer cell death. The CRISPR knockout of MUC4 in PC cells showed increased expression of cleaved caspase-3 after T-cell CM treatment, indicating that MUC4 plays a protective role against the T-cell CM-mediated killing. Subcutaneous implantation of MUC4 proficient and deficient PC cells on the flanks of C57BL/6 immunocompetent mice demonstrated a significantly higher infiltration of CD3 positive and cytotoxic T-cell infiltration in the MUC4 deficient tumors, resulting in significantly lower tumor weight. The PanCancer immune profiling also showed that depletion of Muc4 increases the T-cells and cytotoxic T-cell signaling scores in the KPMC tumor tissues compared to KPC. Conclusion: T-cell secretome induces MUC4 expression in pancreatic cancer cells, and the increased MUC4 expression reduces T-cell infiltration and protects PC cells against T-cell-mediated killing. Citation Format: Xiaoqi Li, Imran Khan, Rachel Kehrberg, Zahraa Wajih Alsafwani, Rakesh Bhatia, Sushil Kumar, Surinder K. Batra. Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response [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 1594.

Abstract 1188: Identification of CXCL10-inducing chemotherapy/targeted therapy combinations for PD-1 blockade sensitization in “cold” triple negative breast cancer

Abstract Metastatic triple-negative breast cancer (mTNBC) currently has the poorest prognosis among subtypes, with limited treatment options due to molecular characteristics that render conventional targeted therapies ineffective. Immunotherapy, specifically anti-PD-(L)1 (ICIs), has revolutionized certain cancer treatments, but its efficacy in mTNBC has been disappointing. To enhance tumor responses to ICIs, combining them with immunogenic chemotherapy has been explored. Studies like IMpassion130 and KEYNOTE 522 indicate benefits of chemo-immunotherapy based on taxanes, especially in PD-L1+ tumors. PD-L1 expression often correlates with TILs infiltration, particularly cytotoxic T lymphocytes (CTLs), indicating an activated immune response in the tumor. However, tumors with poor CTL infiltration, known as "Cold" phenotype, are often associated with a lack of induction of CXCR3-associated chemokines (CXCL9, 10, and 11), crucial for CTLs recruitment in the tumor microenvironment. The limited efficacy of chemo-immunotherapy in PD-L1-negative tumors may stem from chemotherapy's inability to induce immunogenic cancer cell death, CXCR3-associated chemokines, and CTL recruitment. Internal data from a 4T1 TNBC murine model demonstrated that the taxane paclitaxel failed to induce CXCL10, CTL recruitment, and sensitization to PD-L1 blockade. The preclinical research project aimed to identify a combination therapy, inspired by current clinical practices, capable of inducing CXCL10 expression and restoring CTL recruitment in the 4T1 "cold" mTNBC model. We initially screened standard TNBC chemotherapies and selected carboplatin for its in vitro potential to induce CXCL10 production and its clinical use in association with ICIs in the TNBC. Combining carboplatin with a library of over 400 targeted therapies, we identified histone deacetylase (HDAC) inhibitors as significantly enhancing CXCL10 secretion. In vivo, this combination demonstrated therapeutic additivity, increasing immune recruitment, particularly CD8+ T lymphocytes, with the therapeutic effect reduced by CXCR3 blockade. Mechanistic exploration in vitro revealed that the combination activated the type I interferon pathway, inducing CXCL10 secretion. In vivo confirmation validated the role of the type I interferon pathway and CXCL10 in the combination's efficacy. This data outlines a chemotherapy and HDAC inhibitor combination capable of inducing CTL recruitment via increased CXCL10 production. Future project phases will focus on understanding HDACi's mechanism of action on CXCL10 production and the combination's potential to sensitize tumors to ICIs. Overall, this project underscores the role of epigenetic modifications in amplifying carboplatin-induced CXCL10 secretion, potentially converting "cold" ICI-resistant tumors into a "hot" phenotype. Citation Format: Laura Kalfeist, Stacy Petit, Loick Galland, Cyriane Poirrier, Romain Aucagne, François Ghiringhelli, Sylvain Ladoire, Emeric Limagne. Identification of CXCL10-inducing chemotherapy/targeted therapy combinations for PD-1 blockade sensitization in “cold” triple negative breast 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 1188.

Abstract 716: Depletion of stromal cells remodels tumor microenvironment and potentiates immune checkpoint blockade in pancreatic cancer

Abstract During carcinoma progression, mesenchymal stromal cells (MSCs) become recruited to tumors and contribute to the pool of cancer-associated fibroblasts (CAFs). Sub-populations of CAFs, have diverse and incompletely understood effects on disease progression and resistance to therapy. Adipose stromal cells (ASCs), the MSCs from fat tissue increasingly recruited by carcinomas in the context of obesity, have been shown to support cancer progression. Here, the roles of perivascular CAFs expressing platelet-derived growth factor receptor beta (Pdgfrb) and of CAFs expressing non-glycanated decorin (ngDCN), a marker of ASCs, were analyzed. We used mice orthotopically grafted with pancreatic ductal adenocarcinoma (KPC) cells. Ablation of cells expressing thymidine kinase under the control of Pdgfrb promoter with ganciclovir resulted in suppression of primary pancreatic tumor growth. However, ablation Pdgfrb+ cells induced spontaneous metastases to the liver. For depletion of ngDCN+ cells, we used a hunter-killer peptide D-CAN, which has been previously reported to induce apoptosis of ASCs and CAFs in mouse models. Here, D-CAN also had a negative effect on pancreatic tumor growth, however, there was no significant effect on metastatic dissemination. Single cell RNA sequencing of tumors demonstrated that targeting of Pdgfrb+ and ngDCN+ stromal cells had distinct effects on sub-populations of CAFs. Endothelial cell and cancer cell survival was decreased by depletion of either Pdgfrb+ or ngDCN+ stroma. However, pathway analysis revealed that depletion of Pdgfrb+ and ngDCN+ stromal cells has different effects on the markers of cancer cell aggressiveness. D-CAN treatment, and to a less extent Pdgfrb+ cell ablation, suppressed macrophage M2 polarization and increased infiltration of cytotoxic T-lymphocytes. This observation, confirmed in the MyCap graft model of prostate cancer, suggested that ablation of ngDCN+ stroma should synergize with immune checkpoint inhibitors. We tested the effect of D-CAN on the efficacy of anti-PDL1 antibody in the orthotopic KPC model. Compared to D-CAN and anti-PDL1 antibody alone, combination treatment had a synergistic effect on tumor growth. Importantly, liver metastases were suppressed by the D-CAN/anti-PDL1 antibody combination, but not by individual agents. We conclude that improved approaches to target mesenchymal stroma in tumors may be effective in combination with immunotherapy. Citation Format: Joseph Rupert, Alexes Daquinag, Lingyi Cai, Yulin Dai, Zhongming Zhao, Dimitris Anastassiou, Mikhail Kolonin. Depletion of stromal cells remodels tumor microenvironment and potentiates immune checkpoint blockade in pancreatic 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 716.

Abstract 6554: Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Repression of the MHC-I and reduced tumor infiltration of cytotoxic T-lymphocytes is often associated with immunotherapy resistance in SCLC. Lurbinectedin is FDA-approved as a second-line treatment for SCLC and we previously showed lurbinectedin as an effective therapeutic strategy in SCLC preclinical models. There are ongoing clinical trials combining immune checkpoint blockade with lurbinectedin in ES-SCLC. However, there is no insight into the effect of lurbinectedin on the immune microenvironment in SCLC. In this study, we evaluated the effect of lurbinectedin on the immune microenvironment and the anti-tumor effect with or without PD-L1 blockade. Results: To determine the effect of lurbinectedin in SCLC models, we treated immunocompetent flank RPP (Rb1, Trp53, and p130) and RPM (Rb1, Trp53, and MYCT58A) tumor-bearing mice with lurbinectedin (0.2 mg/kg, 1/7days) and/or anti-PD-L1 (300µg, 1/7days) for 3 weeks. Mice treated with anti-PD-L1 alone showed no anti-tumor response, and single-agent lurbinectedin caused a delay in tumor growth. However, 6 of the 10 mice treated with the combination of lurbinectedin and anti-PD-L1, had a complete response (100% reduction) and the other 4 had 90% tumor regression. Tumors were resected and analyzed by multicolor flow cytometry for changes in tumor-infiltrating lymphocytes (TILs). Furthermore, analysis of immune cells infiltrating into the tumors post- treatment showed significant induction of cytotoxic T-cells and a reduction of exhausted and regulatory T cells in the combination treatment arm. Similarly, pro-inflammatory M1 type macrophages and dendritic cells were increased, while immunosuppressive M2 type macrophages and MDSC cells were decreased. These effects are consistent with data showing that lurbinectedin treatment leads to an activation of the cGAS/STING pathway, type I/II interferons (IFNα/β), and pro-inflammatory chemokines (CCL5, CXCL10) in tumors. Interestingly, lurbinectedin treatment led to significant upregulation of mRNA and surface expression of MHC class-I genes (HLA-A/B/C) in vitro and in vivo. Finally, bulk RNA sequencing as well as RT-PCR of pre- and post-lurbinectedin treatment demonstrated an increase in DAMPs associated with immunogenic cell death. Conclusion: We provide the first mechanistic insight into the lurbinectedin-induced multimodal immune modulation in SCLC leading to a dramatic anti-tumor activity accompanied by the establishment of a strong anti-tumor immune microenvironment. Since lurbinectedin is already approved as a second-line agent in SCLC, our preclinical data provide a strong rationale for combining this regimen with inhibitors of the PD-L1 pathway and also highlight the immune subsets that the combination targets. Citation Format: Subhamoy Chakraborty, Yazhini Mahendravarman, Utsav Sen, Avisek Banerjee, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung 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 6554.

Abstract 6539: RNAi mediated silencing of STAT3 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors

Abstract Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical murine tumor models, chemically stabilized acetylated small-interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types in TME and tumor-draining lymph nodes (TDLN), reduced STAT3 protein levels, and increased cytotoxic T-cell infiltration. In murine models of CPI-resistant tumors, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced and resulted in tumor regressions in combination with CPIs. Our findings suggest that targeting STAT3 directly and reducing STAT3 protein in the suppressive immune cell populations in the TME and TDLN may overcome some of the current limitations of immunotherapy and be of therapeutic benefit especially for cancer patients who failed to respond to standard immunotherapy treatments alone. A human active STAT3 conjugate is currently in Phase 1 clinical trial for treatment of immunotherapy refractory cancer types (NCT06098651). Citation Format: Shanthi Ganesh, Minju Kim, Jenny Lee, Xudong Feng, Harini Sivagurunatha Krishnan, Rene Rijnbrand, Amber Beaudry, Jennifer A. Lockridge, Vinita Uttamsingh, John Hanrahan. RNAi mediated silencing of STAT3 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors [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 6539.