Cytotoxic Immune Cells Research Articles

Identification and validation of long noncoding RNA AC083900.1 and RP11-283C24.1 for prediction of progression of osteosarcoma

BackgroundThe role of cuproptosis, an emerging cell death pathway that makes a remarkable contribution to tumor progression, remains elusive in osteosarcoma (OS), in addition to its regulator, including long-no-coding RNAs (lncRNAs) that are also a critical factor for fueling OS. MethodsTranscriptome and clinical data from 70 normal human bone tissue samples and 84 frozen clinical osteosarcoma samples were included in this study. Cuproptosis-associated lncRNAs (CRlncs) were identified through differential expression and co-expression analyses. Univariate Cox regression was performed to screen for prognostic lncRNAs, then we used least absolute shrinkage and selection operator regression to distinguish prognosis-related CRlncs (AC083900.1 and RP11-283C24.1) for modeling the CRlncs prognostic signature (CLPS) by multivariate Cox regression using the stepwise method. CLPS performance was tested by independent prognostic analyses, survival curve and receiver operating characteristic (ROC) curve. In addition, the molecular and immune mechanisms that underlie the unfavorable prognosis of CLPS-identified high-risk group were elucidated. ResultAC083900.1 and RP11–283C24.1 have been identified as the most important CRlncs for OS progression (hazard ratio: 3.498 and 2.724, respectively), and the derived CLPS demonstrated outstanding performance for the prediction of OS prognosis (AUC of 0.799 and 0.778 in the training and test sets, both adj-p < 0.05 in survival curve). As was anticipated, CLPS also outperformed a recent clinical prognostic approach that only achieved an AUC of 0.682 [metastasis]. It is notable that AC083900.1 progressed OS metastasis, evidenced by its high expression in metastatic OS, its high correlation to metastasis-related genes, and its high AUC of 0.683 for the prediction of metastasis. Mechanistically, AC083900.1 and RP11–283C24.1 dysregulated many critical biological processes regarding humoral immune response, immunoglobulin complex, etc.; while reducing the infiltration of many cytotoxic immune cells (B-cells, TIL, neutrophils, etc.). It is encouraging that BMS-509744 and KIN001–135 demonstrated high therapeutic implications for CLPS-identified high-risk OS, and the low-risk counterpart was sensitive to SB-216763. Quantitative RT-PCR analysis showed that both AC083900.1 and RP11-283C24.1 were significantly upregulated in different osteosarcoma cell lines. ConclusionThis study elucidated the roles and mechanisms of AC083900.1 and RP11-283C24.1 in the development of OS, fostering a reliable prognostic approach and treatment for OS patients.

Nanoparticle-enhanced proton beam immunoradiotherapy drives immune activation and durable tumor rejection.

The combination of radiation therapy (RT) and immunotherapy has emerged as a promising treatment option in oncology. Historically, x-ray radiation (XRT) has been the most commonly used form of RT. However, proton beam therapy (PBT) is gaining recognition as a viable alternative, as it has been shown to produce similar outcomes to XRT while minimizing off-target effects. The effects of PBT on the antitumor immune response have only just begun to be described, and to our knowledge no studies to date have examined the effect of PBT as part of a combinatorial immunoradiotherapeutic strategy. Here, using a 2-tumor model of lung cancer in mice, we show that PBT in tandem with an anti-PD1 antibody substantially reduced growth in both irradiated and unirradiated tumors. This was accompanied by robust activation of the immune response, as evidenced by whole-tumor and single-cell RNA sequencing showing upregulation of a multitude of immune-related transcripts. This response was further significantly enhanced by the injection of the tumor to be irradiated with NBTXR3 nanoparticles. Tumors of mice treated with the triple combination exhibited increased infiltration and activation of cytotoxic immune cells. This triple combination eradicated both tumors in 37.5% of the treated mice and showed robust long-term immunity to cancer.

Bakuchiol inhibits lung cancer by modulating tumor microenvironment and the expression of PD-L1.

Immune checkpoint therapy is an emerging frontier in cancer therapy. With the aim to develop an efficient herb derived compound to facilitate immune checkpoint therapy, here we investigate if a herb-derived compound, Bakuchiol (BAK), can be used to treat lung cancer and elucidate if BAK could serve as a PD-L1 regulator. To this end, a murine lung cancer model was established by subcutaneously inoculating murine Lewis lung carcinoma (LLC) cells. BAK of 5 to 40 mg/kg was used for treatment in vivo for 15 days. On Day 15, the population of CD4+ and CD8+ T cells, Treg cells. BAK could effectively inhibit tumor growth by starting treatment either on Day 0 or 6 after tumor inoculation at doses of 5-40 mg/kg. BAK treatment increased the population of cytotoxic immune cells (i.e., CD8+ T cells, and M1 macrophages), meanwhile decreasing pro-tumor immune cells (i.e., CD3+ T cells, Treg cells, and M2 macrophages). Anti-inflammatory cytokines, including IL1β, IL2, IFNγ, TNF-α, IL4 and IL10 were upregulated by BAK. PD-L1 expression in the tumor was also lowered by BAK. AKT and STAT3 signaling were inhibited by BAK. BAK is an efficient agent in reducing LLC tumor growth. These data support the potential of BAK as a new drug for treating lung cancer by serving as a PD-L1 inhibitor that suppresses the activation of AKT and STAT3.

Aerobic Exercise Alters the Melanoma Microenvironment and Modulates ERK5 S496 Phosphorylation.

Exercise changes the tumor microenvironment by remodeling blood vessels and increasing infiltration by cytotoxic immune cells. The mechanisms driving these changes remain unclear. Herein, we demonstrate that exercise normalizes tumor vasculature and upregulates endothelial expression of VCAM1 in YUMMER 1.7 and B16F10 murine models of melanoma but differentially regulates tumor growth, hypoxia, and the immune response. We found that exercise suppressed tumor growth and increased CD8+ T-cell infiltration in YUMMER but not in B16F10 tumors. Single-cell RNA sequencing and flow cytometry revealed exercise modulated the number and phenotype of tumor-infiltrating CD8+ T cells and myeloid cells. Specifically, exercise caused a phenotypic shift in the tumor-associated macrophage population and increased the expression of MHC class II transcripts. We further demonstrated that ERK5 S496A knock-in mice, which are phosphorylation deficient at the S496 residue, "mimicked" the exercise effect when unexercised, yet when exercised, these mice displayed a reversal in the effect of exercise on tumor growth and macrophage polarization compared with wild-type mice. Taken together, our results reveal tumor-specific differences in the immune response to exercise and show that ERK5 signaling via the S496 residue plays a crucial role in exercise-induced tumor microenvironment changes. See related Spotlight by Betof Warner, p. 1158.

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers. We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types. This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Phase 1/2 dose escalation and dose expansion study of TransCon IL-2 β/γ alone or in combination with pembrolizumab: Initial results from dose escalation.

e14597 Background: TransCon IL-2 β/γ is a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog (IL-2 β/γ) designed to optimally address the drawbacks of interleukin-2 (IL-2) therapy. IL-2 β/γ was created by permanently attaching a 5 kDa mPEG in the IL-2Rα binding site. To improve the pharmacokinetic (PK) properties, IL-2 β/γ was transiently attached to a 40 kDa mPEG carrier via a TransCon linker creating a prodrug, TransCon IL-2 β/γ. Under physiological conditions, TransCon IL-2 β/γ releases active IL-2 β/γ from the carrier via autocleavage in a controlled manner. The associated long effective half-life and sustained IL-2 β/γ exposure results in robust activation of IL-Rβ/γ+ cytotoxic immune cells, at a lower Cmax compared to IL-2 therapy. The primary objectives of the IL-Believe trial are to evaluate safety and tolerability, and to define the maximum tolerated dose and recommended Phase 2 dose of TransCon IL-2 β/γ alone or in combination with pembrolizumab. Methods: In dose escalation (3+3 design), patients aged ≥18 with advanced solid tumors receive TransCon IL-2 β/γ as monotherapy or in combination with pembrolizumab with IV dosing every 3 weeks. Disease is assessed every 9 weeks using RECIST 1.1. Safety, PK, and pharmacodynamics (PD) are evaluated. Results: As of 25 Jan 2023, 27 patients were treated in dose escalation: 18 in monotherapy (Dose Levels (DL) 1-4) and 9 in combination therapy (DL1, DL2). The most common tumor types in monotherapy and in combination therapy were head &amp; neck (19%), colorectal (11%) and endometrial (11%). Patients received a median of 3 prior lines of therapy in monotherapy. The most common Treatment Related Adverse Events (TRAEs) in monotherapy and in combination therapy were pyrexia (30%), Cytokine Release Syndrome (CRS) (26%) and fatigue (22%). Grade 3 (G3) TRAEs in monotherapy were anemia (17%), CRS (6%), thrombocytopenia (6%), hypoxia (6%), all reported at DL4. 1 Dose Limiting Toxicity of G3 CRS was reported at DL4. Preliminary PK results showed systemic drug exposure of TransCon IL-2 β/γ with a half-life of ~ 35 hours and no PK interaction with pembrolizumab was observed. PD data were consistent with sustained activation and dose dependent expansion of cytotoxic effector cells, in comparison, Tregs were only expanded minimally. Absolute lymphocyte counts increased in a dose dependent manner while eosinophils remained low. Prolonged stable disease ( &gt; 32 weeks) was observed in one pancreatic cancer patient on monotherapy. Conclusions: TransCon IL-2 β/γ is generally well-tolerated as monotherapy and in combination with pembrolizumab. The pharmacodynamics data confirm a durable activation and expansion of IL-2Rβ/γ+ cytotoxic immune cells over IL-2Rα+ eosinophils and Tregs. Clinical trial information: NCT05081609 .

Immune features of ovarian tumors with a good response to neo-adjuvant chemotherapy in combination with an anti-PD-L1 alone or with an anti-CTLA4: Data from the randomized IneOV trial (a GINECO study).

5586 Background: We have shown that chemotherapy may have immunomodulatory properties and prime the tumor microenvironment (TME) by recruiting cytotoxic immune cells. We previously reported that neoadjuvant chemotherapy (NACT) associated with Durvalumab (D) +/- Tremelimumab (T) resulted in encouraging complete resection (70%) and complete pathological response (18%) rates in patients with unresectable ovarian cancer. Here, we aimed to 1) characterize the immune TME at baseline of responders compared to non-responders and 2) describe changes in immune TME in paired tumor samples at diagnosis and after 3 cycles of NACT with D +/- T in the randomized IneOV trial. Methods: Tumor samples from IneOV trial (55 at diagnosis, 40 after treatment) were analyzed for CD3+, CD68+ and CD20+ by multiplexed immunohistochemistry. Stromal and intra-epithelial TILs were scored as percentage of positive surface. Patients were classified as good pathological responders (pR) (CRS3) or pathological non-responders (pNR) (CRS1 or 2). Intra-tumoral T cell infiltration was described as High or Low (intraepithelial (ie)CD3+ &gt; median vs &lt; , respectively). Non parametric statistical tests were used. We evaluated the correlation of the markers with each others (Spearman test) and with pathological response rate (Mann-Withney test). Results: At diagnosis, in the intra-epithelial compartment of the TME, the most abundant cells were macrophages (median ieCD68+ = 3,4) compared to T (ieCD3+ = 1.1), B (ieCD20+ ND) ANOVA p-value = 0.002 and &lt; 0.0001 respectively . In individual tumors, infiltration by the 3 different immune subsets was poorly correlated suggesting immune TME heterogeneity in OC. Pathological response was evaluable in 64 patients: 19 pR and 45 pNR. At diagnosis, CD3+ (p = 0.02) and CD68+ (p = 0.006) infiltration was significantly higher in pR tumors compared to pNR. Most of pR (76%) had high intra-tumoral CD3+ infiltration versus 39% in pNR (p = 0.011). After treatment, tumors with pR showed significantly greater CD3+ intra-epithelial infiltration compared to pNR (p = 0.047). Conclusions: Our results suggest that good pathological response to NACT +D+/-T is associated with high levels of both T cells and macrophages in iTME at baseline, and increased intratumoral T cell infiltration post-treatment. Work is ongoing to further characterize the iTME, especially in pNR to identify other strategies to enhance the anti-tumor immune response. Clinical trial information: NCT03249142 .

Multi-omics analysis of atezolizumab plus bevacizumab in patients with unresectable or metastatic mucosal melanoma.

e21539 Background: A multicenter, open-label, single-arm phase II study has demonstrated that atezolizumab plus bevacizumab showed promising efficacy and manageable safety in patients with advanced mucosal melanoma, with an ORR of 45.0%. Here, we report the multi-omics analysis results with the aim of identifying potential biomarkers of response to this combination therapy. Methods: Patients with available samples were included in this study. The FFPE tissue was performed for DNA and RNA co-extraction and underwent whole exome sequencing (WES) and transcriptome sequencing. The responses were classified as complete response (CR) and partial response (PR) for responders and stable disease (SD) and progressive disease (PD) for non-responders per RECIST v1.1. Mutational signature, SNVs clonality, copy-number variations, differential expression genes, GSEA, and the immune microenvironment analysis were performed and analyzed for correlation with clinical benefits. Results: A total of 29 patients with FFPE samples underwent WES and/or transcriptome sequencing, including 1 CR, 15 PR, 9 SD, and 4 PD. As of January 11, 2023, the median follow-up time was 31 months (range, 5.0 to 37.0 months). Compared with non-reponders, responders had a significantly longer PFS (median 14.0, 95% CI 9.1 to 16.9 months vs 2.0, 95% CI 1.5 to 2.5 months, P&lt; 0.001) and OS (median 35.0, 95% CI 20.4 to 49.6 months vs 14.0, 95% CI 5.5 to 22.5 months, P =0.005). Of the patients who underwent WES analysis, TMB and TNB were not associated with response, PFS or OS; but 53.3% (8/15) of responders harbored NRAS/PKHDL1 mutation, whereas only 16.7% (2/12) of non-reponders harbored NRAS/PKHDL1 mutation ( P= 0.112). Transcriptome analysis revealed that compared with non-reponders, responders displayed more significant angiogenesis, T cell cytotoxicity and interferon gamma response gene expression signatures. CIBERSORT analysis showed that responsders had higher proportions of cytotoxic immune cells, and non-responders had higher a higher proportion of B cells. The expression of CD274 was not associated with the response, and the expression of CD276 was significantly higher in responders than that in non-responders ( P =0.014). Weighted gene co-expression network analysis showed that the high expression of genes associated with the p38 MAPK pathway presented in cold tumor environments ( P =0.052) and was negatively correlated to the PFS ( P&lt; 0.001) and OS ( P&lt; 0.001). Conclusions: These molecular profiles indicated that the inflammatory cell infiltration and tumor angiogenic status was associated with response to anti-PD-L1 and anti-VEGF combination treatments, and targeting p38 MAPK pathway may further improve the survival of patients with mucosal melanoma.

Impact of Aneuploidy and Chromosome 9p Loss on Tumor Immune Microenvironment and Immune Checkpoint Inhibitor Efficacy in NSCLC

IntroductionAlthough gene-level copy number alterations have been studied as a potential biomarker of immunotherapy efficacy in NSCLC, the impact of aneuploidy burden and chromosomal arm-level events on immune checkpoint inhibitor (ICI) efficacy in NSCLC is uncertain. MethodsPatients who received programmed cell death protein 1 or programmed death-ligand 1 (PD-L1) inhibitor at two academic centers were included. Across all 22 chromosomes analyzed, an arm was considered altered if at least 70% of its territory was either gained or deleted. Among nonsquamous NSCLCs which underwent targeted next-generation sequencing, we retrospectively quantified aneuploidy using the adjusted fraction of chromosomal arm alterations (FAA), defined as the number of altered chromosome arms divided by the number of chromosome arms assessed, adjusted for tumor purity. ResultsAmong 2293 nonsquamous NSCLCs identified, the median FAA increased with more advanced cancer stage and decreased with higher PD-L1 tumor proportion score (TPS) levels (median FAA in TPS < 1%: 0.09, TPS 1%–49%: 0.08, TPS ≥ 50%: 0.05, p < 0.0001). There was a very weak correlation between FAA and tumor mutational burden when taken as continuous variables (R: 0.07, p = 0.0005). A total of 765 advanced nonsquamous NSCLCs with available FAA values were treated with ICIs. With decreasing FAA tertiles, there was a progressive improvement in objective response rate (ORR 15.1% in upper tertile versus 23.2% in middle tertile versus 28.4% in lowest tertile, p = 0.001), median progression-free survival (mPFS 2.5 versus 3.3 versus 4.1 mo, p < 0.0001), and median overall survival (mOS 12.5 versus 13.9 versus 16.4 mo, p = 0.006), respectively. In the arm-level enrichment analysis, chromosome 9p loss (OR = 0.22, Q = 0.0002) and chromosome 1q gain (OR = 0.43, Q = 0.002) were significantly enriched in ICI nonresponders after false discovery rate adjustment. Compared with NSCLCs without chromosome 9p loss (n = 452), those with 9p loss (n = 154) had a lower ORR (28.1% versus 7.8%, p < 0.0001), a shorter mPFS (4.1 versus 2.3 mo, p < 0.0001), and a shorter mOS (18.0 versus 9.6 mo, p < 0.0001) to immunotherapy. In addition, among NSCLCs with high PD-L1 expression (TPS ≥ 50%), chromosome 9p loss was associated with lower ORR (43% versus 6%, p < 0.0001), shorter mPFS (6.4 versus 2.6 mo, p = 0.0006), and shorter mOS (30.2 versus 14.3 mo, p = 0.0008) to immunotherapy compared with NSCLCs without 9p loss. In multivariable analysis, adjusting for key variables including FAA, chromosome 9p loss, but not 1q gain, retained a significant impact on ORR (hazard ratio [HR] = 0.25, p < 0.001), mPFS (HR = 1.49, p = 0.001), and mOS (HR = 1.47, p = 0.003). Multiplexed immunofluorescence and computational deconvolution of RNA sequencing data revealed that tumors with either high FAA levels or chromosome 9p loss had significantly fewer tumor-associated cytotoxic immune cells. ConclusionsNonsquamous NSCLCs with high aneuploidy and chromosome 9p loss have a distinct tumor immune microenvironment and less favorable outcomes to ICIs.

Mesenchymal stem cell-derived microRNAs: Friends or foes of tumor cells?

Mesenchymal stem cell (MSC)-dependent biological effects in the tumor microenvironment mainly rely on the activity of MSC-sourced microRNAs (MSC-miRNAs) which modulate protein synthesis in target tumor cells, endothelial cells and tumor-infiltrated immune cells, regulating their phenotype and function. Several MSC-sourced miRNAs (miR-221, miR-23b, miR-21-5p, miR-222/223, miR-15a miR-424, miR-30b, miR-30c) possess tumor-promoting properties and are able to enhance viability, invasiveness and metastatic potential of malignant cells, induce proliferation and sprouting of tumor endothelial cells and suppress effector functions of cytotoxic tumor-infiltrated immune cells, crucially contributing to the rapid growth and progression of tumor tissue. On the contrary, MSCs also produce "anti-tumorigenic" miRNAs (miR-100, miR-222-3p, miR-146b miR-302a, miR-338-5p, miR-100-5p and miR-1246) which suppress tumor growth and progression by: Up-regulating expression of chemoresistance-related genes in tumor cells, by suppressing neo-angiogenesis and by inducing generation of tumorotoxic phenotypes in tumor-infiltrated lymphocytes. In this review article, we summarize the current knowledge about molecular mechanisms that are responsible for MSC-miRNA-dependent alterations of intracellular signaling in tumor and immune cells and we discuss different insights regarding the therapeutic potential of MSC-derived miRNAs in cancer treatment.

Prognostic association of immunoproteasome expression in solid tumours is governed by the immediate immune environment.

Induction of immunoproteasome (IP) expression in tumour cells can enhance antigen presentation and immunogenicity. Recently, overexpression of IP genes has been associated with better prognosis and response to immune checkpoint blockade (ICB) therapies in melanoma. However, the extent of this association in other solid tumours and how that is influenced by tumour cell-intrinsic and cell-extrinsic factors remains unclear. Here, we address this by exploring the gene expression patterns from available bulk and single-cell transcriptomic data of primary tumours. We find that tumours with high IP expression exhibit cytotoxic immune cell infiltration and upregulation of IFN-γ and TNF-α pathways in tumour cells. However, the association of IP expression with overall survival (TCGA cohort) and response to ICB therapy (non-TCGA cohorts) is tumour-type specific (better in non-small-cell lung, breast, bladder, and thymus; and worse in glioma and renal) and is greatly influenced by pro- or anti-tumorigenic immune cell infiltration patterns. This emphasises the need for considering immune cell infiltration patterns, along with IP expression, as a prognostic biomarker to predict overall survival or response to ICB therapies in solid tumours, besides melanoma.

Enabling Chemo-Immunotherapy with HIFU in Canine Cancer Patients.

High intensity focused ultrasound (HIFU) is a promising non-invasive technique for treating solid tumors using thermal and histotripsy-based mechanical ablation. However, its clinical significance in different tumor types is not fully understood. To assess its therapeutic efficacy and immunomodulatory properties, we compared HIFU thermal ablation and histotripsy ablation in dogs with spontaneous tumors. We also evaluated the ability of non-ablative HIFU-based mild hyperthermia (40-45 ºC) to improve Doxorubicin delivery and immunomodulation. Our results showed that HIFU thermal ablation induced tumor remission in the majority of treated patients over 60days, while histotripsy achieved partial response to stable disease persistence. The adverse effects of thermal ablation were minor to moderate, while histotripsy exposures were relatively well-tolerated. Furthermore, we observed a correlation between HIFU-therapeutic response and serum anti-tumor cytokine profiles and the presence of functionally active cytotoxic immune cells in patients. Similarly, Doxorubicin-treated patients showed improved drug delivery, efficacy, and anti-tumor immune responses with HIFU hyperthermia. In conclusion, our study demonstrates that depending on the tumor type and treatment parameters, HIFU treatments can enable tumor growth control, immune activation, and chemotherapy in veterinary patient. These findings have significant clinical implications and highlight the potential of HIFU as a promising cancer treatment approach.

Heterogeneity in the immune microenvironment of bone metastasis in driver-positive non-small cell lung cancer.

Mutations in epidermal growth factor receptor and anaplastic lymphoma kinase are common driver events in non-small cell lung cancer (NSCLC), which are associated with a high frequency of bone metastases (BMs). While the bone marrow represents a specialized immune microenvironment, the immune repertoire of BMs remains unknown. Considering the higher incidence of BMs in driver gene-positive NSCLCs, and the unique biology of the bone, herein, we assessed the infiltrating immune cells and T cell receptor (TCR) profile of BMs in driver-positive NSCLCs. Immune profile of BMs in driver gene-positive NSCLC were assessed in 10 patients, where 6 had driver gene-positive mutation. TCR and bulk RNA sequencing were performed on malignant bone samples. The diversity and clonality of the TCR repertoire were analyzed. The cellular components were inferred from bulk gene expression profiles computationally by CIBERSORT. Although BMs were generally regarded as immune-cold tumors, immune cell composition analyses showed co-existence of cytotoxic and suppressor immune cells in driver-positive BM samples, as compared to primary lung. Analysis of the TCR repertoire indicated a trend of higher diversity and similar clonality in the driver-positive compared with the driver-negative subsets. In addition, we identified two cases that showed the opposite response to immune checkpoint blockade. A comparison of these two patients' BM samples showed more highly amplified clones, fewer M2 macrophages and more activated natural killer cells in the responder. In summary, BMs in NSCLC are heterogeneous in their immune microenvironment, which might be related to differential clinical outcomes to immune checkpoint blockade.

Abstract 4786: Imipridones induce lipid peroxidation and oxidative stress in gastrointestinal malignancies

Abstract Colorectal and pancreatic cancer collectively comprise 20% of all cancer-related deaths in the United States. Despite therapeutic advancements, the current standard of care often results in significant toxicity. Therefore, there is an urgent need to develop more efficacious novel therapies. The imipridones, ONC201/TIC10, ONC206, and ONC212, are novel small-molecules that bind mitochondrial ClpP and induce activation of the integrated stress response (ISR), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling, and oxidative stress. Each has previously demonstrated antitumor effects in a variety of preclinical in vitro and in vivo cancer subtypes, including colorectal cancer, with limited toxicity. Reactive oxidation species (ROS) are natural biproducts of cellular oxidative metabolism and play important roles in the modulation of cell death pathways, differentiation, and immune response. Using varying doses of ONC201, ONC206, and ONC212, we observed increased lipid peroxidation in several colorectal and pancreatic cancer cell lines using the C-11 BODIPY assay which measures lipid-specific peroxides. Lipid peroxidation was reversed by the ferroptosis inhibitor liproxstatin-1, suggesting that in addition to inducing apoptosis, imipridones may be involved in ferroptosis induction. Future directions include mechanistic work, investigation of the effect of imipridone-induced lipid peroxidation on immune cell cytotoxicity, and understanding the relative importance of lipid peroxidation in the clinical efficacy of imipridones. Citation Format: Arielle De La Cruz, Praveen R. Srinivasan, Andrew George, Varun V. Prabhu, Maximilian P. Pinho-Schwermann, Wafik S. El-Deiry. Imipridones induce lipid peroxidation and oxidative stress in gastrointestinal malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4786.

Abstract 3230: Tumor microenvironment modulation by immunotherapy sensitizes solid tumors to radiation

Abstract Radiation therapy (RT) has been prevalently implemented to treat cancer in clinical settings by inducing DNA damage in tumor cells. However, tumor microenvironmental factors such as severe hypoxic conditions and vascular anomalies attenuate the therapeutic efficacy of RT, thus resulting in unsatisfactory outcomes in solid tumors. Recently, Immune checkpoint blockade (ICB) has improved immune recognition to tumor cells and normalized tumor vasculature to diminish hypoxia in solid tumors. Herein, we suggest that RT combined with ICB synergistically alters tumor microenvironments, thus facilitating the recruitment of cytotoxic immune cells into tumors as well as sensitizing radiation effectiveness. RT in combination with ICB not only normalized tumor vasculature to reduce hypoxia, but also increased anti-tumor effects from immune-mediated toxicity and DNA damage-mediated apoptosis in several solid tumor-bearing mouse models to a greater extent than did either monotreatment of RT or ICB. This study will provide mechanistic insight into synergistic effects conferred by RT and ICB as well as a strong preclinical rationale for combining the two therapeutic modalities in human cancers. Citation Format: Minjeong Kang, DaeYong Lee, Yifan Wang, Betty Kim, Wen Jiang. Tumor microenvironment modulation by immunotherapy sensitizes solid tumors to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3230.

Abstract 5166: Intratumoral delivery of mRNA encoding USP6 activates multiple immuno-stimulatory pathways simultaneously and inhibits local and distal tumor growth in murine models

Abstract Advances in immunotherapy have revolutionized cancer treatment, with patients with otherwise incurable disease having long-lasting responses. Unfortunately, deep remissions only occur in a select subset of patients. Cancer progression is common as many current immunotherapies only target one or two pathways, which can quickly lead to tumor resistance. Novel approaches that target several pathways concurrently are therefore needed to promote stronger responses in a wider patient population. Our group has recently discovered that the protein Ubiquitin-specific protease 6 (USP6) upregulates multiple, distinct immunostimulatory pathways. Tumor cell-specific expression of USP6 significantly increases CXCL9, CXCL10, and CCL5 chemokine production that enhances cytotoxic immune cell recruitment. Simultaneously, USP6 also upregulates several ligands and receptors that promote immune cell activation and killing of tumor cells such as CD112, MICA/B, ICAM1, and MHC Class I. In addition, USP6 increases the tumor cell’s sensitivity to immune-derived factors such as Type I and Type II interferons and TRAIL by raising the surface expression of their respective receptors. USP6 is hominid-specific and therefore requires xenografting human tumor cells into athymic, T-cell deficient nude mice. Despite the immunocompromised model, USP6 expression was found to increase immune cell recruitment to the tumor while repolarizing the immune microenvironment toward a more activated, tumor-suppressive state. Strikingly, immune cells in the peripheral blood were also affected with an increase in natural killer cell (NK) activation and a skewing of the myeloid population away from an immunosuppressive phenotype. Our group subsequently demonstrated that a USP6+ tumor was able to inhibit the growth of a USP6− distal tumor, with a concordant increase in NK, macrophage, and dendritic cell activation in the distal USP6− tumor. Given our data, we sought to translate USP6 into a novel immunotherapy. Delivery of mRNA encoding the USP6 protein recapitulates the immunostimulatory effects of USP6 in vitro. Intratumoral (I.T) injection of a lipid nanoparticle (LNP) encapsulating USP6 mRNA significantly inhibited Ewing sarcoma and acute myeloid leukemia growth in athymic mice, despite the model lacking a fully functional immune system. Moreover, I.T injection of USP6 mRNA LNPs inhibited the growth of a non-injected, distal tumor. Altogether, these data support that USP6 is a potentially powerful target and continued development of I.T USP6 mRNA LNPs as a novel immunotherapy. Citation Format: Ian Henrich, Margaret Billingsley, Kanika Jain, Laura Quick, Rob Young, Margaret Chou, Michael Mitchell. Intratumoral delivery of mRNA encoding USP6 activates multiple immuno-stimulatory pathways simultaneously and inhibits local and distal tumor growth in murine models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5166.

Abstract 6548: Bioinformatic characterization of dendritic and T cell subpopulations in the tumor microenvironment across different cancer types by single-cell RNA-Seq analysis

Abstract Although the development of single-cell RNAseq (scRNAseq) has improved the knowledge of the tumor microenvironment (TME), there is not a clear understanding of specific cell subtypes, markers that define these cell populations, nor the interaction between them. Previously, we showed that inflammasome activation is correlated with antitumor responses triggered by immune checkpoint blockade (ICB). This highlights a central role for TMEM176B, mostly expressed in myeloid cells, that impairs the recruitment of cytotoxic immune cells in the TME by inhibiting NLRP3 inflammasome activation. Still, the roles of the different subsets of myeloid and T cells in the TME and to what extent they are connected remain controversial. Here, we used scRNAseq data from human tumors to identify and characterize cDC2, Th17 and exhausted CD8+ T cell subpopulations in cancer. Five scRNAseq data sets of melanoma, basal cell carcinoma, breast, lung, colorectal, and ovary cancer patients were integrated, accounting for 382,019 immune cells in total. We applied and compared different bioinformatic tools for scRNASeq data normalization and data sets integration to find conserved immune cell populations across samples, data sets, and cancer types. Then, we evaluated different parameters using well-established methods for clustering and cell type classification to identify subtypes of DCs and T cells in the integrated data. We combined automatic methods with manual analysis of cluster markers to improve robustness and confidence in the cell populations identified. Three clusters of cDC2 were defined and characterized using different scRNAseq analysis tools for pathway enrichment analysis and transcription factors (TF) activation prediction. We found a subset of inflammatory cDC2 with high gene expression of IL1B and NLRP3, in contrast with another cluster characterized by high expression of TMEM176B and CD14. A third cluster showed an intermediate transcriptomic profile. The TMEM176B+ CD14+ cluster was characterized by the activation of JAK-STAT and hypoxia pathways, and the TF FOXL2, ETS2, and ESR2. The NLRP3+ IL1B+ cluster showed more activation of MAPK, Trail, and PI3K pathways, and the TF CEBPE. Moreover, the activation of TNFα and NFkB signaling pathways was found in both clusters. In addition, a conserved population of Th17 cells in the integrated data was observed. Th17 cells were heterogeneous, making the subpopulations and transcriptome signature difficult to define. Further analysis is being performed to identify Th17 subsets and their potential interactions with the cDC2 subclusters. A robust identification and characterization of these immune cell subsets will build towards our understanding of the interactions between these subpopulations triggered by inflammasome activation within the TME and the mechanisms behind ICB response. Citation Format: Yamil D. Mahmoud, Natalia Rego, Marcela Vilarino, Florencia Veigas, Maria R. Girotti, Juan M. Perez Saez, Gabriel A. Rabinovich, Marcelo Hill. Bioinformatic characterization of dendritic and T cell subpopulations in the tumor microenvironment across different cancer types by single-cell RNA-Seq analysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6548.

Abstract 5120: NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic human NSCLC in a humanized mouse model

Abstract NPRL2/TUSC4 is a potent tumor suppressor gene whose expression is reduced in many cancers including NSCLC. Restoration of NPRL2 expression in cancer cells induces DNA damages which leads to cell cycle arrest and apoptosis. We investigated the antitumor immune responses to NPRL2 gene therapy on anti-PD1 resistant KRAS/STK11 mutant NSCLC in a humanized mouse model. H1299 cells transfected with NPRL2 showed significant inhibition of colony formation after NPRL2 transfection. Humanized mice were generated by transplanting fresh human cord blood derived CD34 stem cells into sub-lethally irradiated NSG mice. The level of engraftment of human CD45, CD3 T, CD19 B, NK cells was verified before tumor implantation. Mice harboring &amp;gt; 25% human CD45 cells were considered humanized. KRAS/STK11 mutant anti-PD1 resistant A549 NSCLC cells were injected intravenously into humanized NSG mice and developed lung metastasis. Metastases were treated with intravenous injection of NPRL2 gene loaded cationic lipid nanoparticles with or without pembrolizumab (anti-PD1). A dramatic antitumor effect was mediated by NPRL2 treatment, whereas pembrolizumab was ineffective. A significant antitumor effect was also found in non-humanized NSG mice, although the effect was greater in humanized mice suggesting that the possible role of antitumor immunity. The antitumor effect of NPRL2 was associated with increased infiltration of human CD45, CD3 T, cytotoxic T, NK cells, and a decreased number of human regulatory T cells (Treg) in tumors. PD1 expressing exhausted CD8 T cells were downregulated in both the NPRL2 and pembrolizumab groups. The number of activated T cells (CD69+CD8+T), effector (EM) and central memory (CM) CD8 T cells were significantly increased by NPRL2 treatment. NPRL2 induced antigen presenting HLA-DR+ dendritic cells. When NPRL2 was combined with pembrolizumab, no synergistic antitumor effect was found in the KRAS/STK11 mutant anti-PD1 insensitive tumors. However, a robust and synergistic antitumor effect was observed in the KRAS wild type, anti-PD1 sensitive H1299 tumors grown in humanized mice treated with NPRL2 + pembrolizumab. Cytotoxic T cells, NK cells, and HLA-DR+ DC were associated with the antitumor effect. DOTAP-NPRL2 was tested in a syngeneic mouse model with LLC2 tumors that are KRAS mutant and anti-PD1 resistant. Consistent with the A549 humanized mouse model, NPRL2 showed a significantly strong antitumor effect whereas anti-PD1 was not effective in this model. The antitumor effect of NPRL2 was again correlated with the upregulation of HLA-DR+ DC, CD11c DC, TILs, NK and downregulation of Treg and myeloid cells in the tumor microenvironment. Taken together, these data suggest that NPRL2 gene therapy induces antitumor activity on KRAS/STK11 mutant anti-PD1 resistant tumors through DC mediated antigen presentation and cytotoxic immune cell activation. Citation Format: Ismail M. Meraz, Mourad Majidi, Renduo Song, Feng Meng, Gao Lihui, Qi Wang, Jing Wang, Elizabeth Shpall, Jack A. Roth. NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic human NSCLC in a humanized mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5120.

Abstract 1270: ECHS1 mediates metabolic disruption in hormone receptor-positive breast tumor microenvironment

Abstract Unchecked hyperadiposity causes systemic metabolic perturbations and subclinical chronic inflammation, promoting hormone receptor positive (HR+) breast cancer. Murine models of high-fat diet-induced obesity have shown alterations in proteins involved in fatty acid binding and mitochondrial beta-oxidation, including enoyl-CoA hydratase short chain 1 (ECHS1), which promote increased uptake of fatty acids by primary tumor cells. This creates a metabolic tug of war between tumor and immune cells in the tumor microenvironment, thereby depriving cytotoxic immune cells of the metabolic reprogramming for anti-tumor functionality. Nonetheless, metabolic gene expression changes in breast tumor microenvironment of obese individuals remain elusive. We hypothesize that increased expression of ECHS1 leads to immune dysregulation in breast tumor microenvironment and increases risk of cancer progression. Proteomic and genomic expression and survival characteristics of ECHS1 in invasive breast cancer was explored in The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium. We the modeled the effects of ECHS1 expression on immune infiltration in the tumor microenvironment using a regression framework. We also performed single cell RNA sequencing analysis for differential expression of ECHS1 in various human breast cells. Additionally, expression level of ECHS1 was validated in silico in adipose tissue to elucidate increased immune dysregulation and risk of progression of breast carcinoma in obese individuals. We found a significantly increased expression of ECHS1 at both RNA and protein levels in HR+ breast cancer (p&amp;lt;0.001 for both), compared to Herceptin 2 receptor-positive or triple negative breast cancer. High expression of ECHS1 in female breast cancer is also associated with significantly decreased survival based on the TCGA data. Further, we found a significantly negative correlation of CD8+ T cells, neutrophils, and macrophages, and a significantly positive correlation of regulatory T cells with ECHS1 expression in the breast tumor microenvironment. We discovered increased expression of ECHS1 in luminal epithelial cells compared to myoepithelial cells based on single cell RNA sequencing. Lastly, high expression of ECHS1 protein expression based on immunohistochemistry was confirmed in human adipocytes. Collectively, our observations support the hypothesis that preferential uptake of free fatty acid through increased expression of ECHS1 in HR+ breast cancer impairs cytotoxic and anti-tumor effects of CD8+ T cells in the tumor microenvironment. Immune dysregulation is further amplified in obese individuals given increased levels of adipose cells and higher ECHS1 expression. Altogether, ECHS1 is a putative biomarker and potential therapeutic target as its downregulation may improve survival in obese patients with HR+ breast cancer. Citation Format: Tina Bharani, Divyansh Agarwal, Estefania Roldan-Vasquez, Jessalyn Ubellacker, Ted A. James. ECHS1 mediates metabolic disruption in hormone receptor-positive breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1270.

Abstract 5874: HSF1 downregulation of CCL5 reduces CD8 T cell trafficking in breast cancer

Abstract Breast cancer is the leading cause of cancer related deaths in women. The presence of cytotoxic immune cells, specifically CD8+ T cells, in breast tumors is associated with better patient outcomes. Our goal is to understand mechanisms that regulate the infiltration of CD8+ T cells. This will in turn, improve the treatment and enhance patient overall survival. Utilizing a novel HSF1 activity gene signature, we observed that HSF1 activity was negatively associated with the presence of CD8+ T cells in breast cancer patients. Both CIBERSORT analysis of TCGA data as well as direct assessment of patient primary specimens demonstrated that patient tumors with high HSF1 activity had lower numbers of CD8+ T cells. To functionally test this relationship, HSF1 was knocked down in 4T1 breast cancer cells and grown orthotopically in BALB/c mice. Tumors with HSF1 knockdown showed lower tumor volumes and increased CD8+ T cell infiltration. We subjected the control and knockdown tumors to single- cell RNA sequencing analysis and found increased immune cell presence specifically CD8+ T cell presence in knockdown tumors. We further tested the effect of CD8+ T cells on the growth of HSF1 knockdown tumors by depleting CD8+ T cells in BALB/c mice. HSF1 knockdown tumors were significantly larger with CD8+ T cell depletion suggesting a functional role for HSF1 to inhibit CD8+ T cell infiltration and protect the tumor from immune-mediated killing. To investigate the mechanism by which HSF1 affects CD8+ T cells, we investigated whether HSF1 affects chemotactic cytokines. We found that loss of HSF1 significantly increased secretion of CCL5, a known chemoattractant for CD8+ T cells. Loss of HSF1 also increased mRNA levels of CCL5, suggesting a transcriptional effect on CCL5. To test the importance of CCL5 in the phenotype of HSF1 knockdown, a transwell migration assay was performed wherein T cells from BALB/c mice were placed in the upper chamber of a transwell chamber and the lower chamber contained conditioned medium from 4T1 cells with knockdown of HSF1 with or without knockdown of CCL5. The lower chamber was subjected to flow cytometry for CD3/CD8 to identify CD8+ T cells and we observed that loss of HSF1 increased migration of CD8+ T cells and this was ablated with additional loss of CCL5. These results suggest CCL5 is a significant mediator for the recruitment of CD8+ T cells when HSF1 is lost in cancer cells. We propose the model whereby high HSF1 activity in breast cancer cells suppresses expression and secretion of the chemo-attractant cytokine CCL5 that ultimately leads to a decrease in CD8+ T cells in breast tumors microenvironment. Citation Format: Curteisha LeeAnn Jacobs, Sakhi Shah, Wen-Cheng Lu, Richard Carpenter, Haimanti Ray, Xin Lu, Sha Cao. HSF1 downregulation of CCL5 reduces CD8 T cell trafficking in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5874.