Modulation Of Tumor Immune Microenvironment Research Articles

Sophora alopecuroide - Taraxacum decoction (STD) inhibits non-small cell lung cancer via inducing ferroptosis and modulating tumor immune microenvironment

The Sophora alopecuroide - Taraxacum Decoction (STD) is a traditional Chinese herbal formulation that has demonstrated significant potential in combating tumors. Despite its apparent effectiveness, the specific mechanisms through which STD exerts its anti-tumor properties remain largely unexplored and are yet to be fully understood. In our study, we provided evidence that STD effectively inhibits cellular growth and movement, as well as halting the cell cycle at the G2/M checkpoint. Furthermore, our pharmacological network analysis indicated that STD might induce cell death through a process known as ferroptosis. This hypothesis was substantiated by observing important biochemical changes associated with ferroptosis, including a decrease in glutathione (GSH) levels, an increase in iron accumulation, and elevated levels of reactive oxygen species (ROS) and lipid peroxidation. Additionally, we noted a significant rise in the expression of pro-ferroptosis genes such as Keap1, Nrf2, and HO-1, further supporting our findings. Significantly, and in line with the in vitro results, STD also showed a strong ability to inhibit tumor growth by inducing ferroptosis in a subcutaneous tumor model. Additionally, STD treatment changed the tumor immune microenvironment (TIME), as seen by an increase in CD107a+ CD8 and NK cells within the tumor. These findings demonstrate that STD induces ferroptosis and alters TIME to combat tumors, suggesting that STD may be a viable alternative treatment for patients with NSCLC.

Circ_BBS9 as an early diagnostic biomarker for lung adenocarcinoma: direct interaction with IFIT3 in the modulation of tumor immune microenvironment.

Introduction: Circular RNAs (circRNAs) have been identified as significant contributors to the development and advancement of cancer. The objective of this study was to examine the expression and clinical implications of circRNA circ_BBS9 in lung adenocarcinoma (LUAD), as well as its potential modes of action. The expression of Circ_BBS9 was examined in tissues and cell lines of LUAD through the utilization of microarray profiling, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. In this study, we assessed the impact of circ_BBS9 on the proliferation of LUAD cells, as well as its influence on ferroptosis and tumor formation. To analyze these effects, we employed CCK-8 assays and ferroptosis assays. The identification of proteins that interact with Circ_BBS9 was achieved through the utilization of RNA pull-down and mass spectrometry techniques. A putative regulatory network comprising circ_BBS9, miR-7150, and IFIT3 was established using bioinformatics study. The investigation also encompassed the examination of the correlation between the expression of IFIT3 and the invasion of immune cells. Circ_BBS9 was significantly downregulated in LUAD tissues and cell lines. Low circ_BBS9 expression correlated with poor prognosis. Functional experiments showed that circ_BBS9 overexpression inhibited LUAD cell proliferation and promoted ferroptosis in vitro and suppressed tumor growth in vivo. Mechanistically, circ_BBS9 was found to directly interact with IFIT3 and regulate its expression by acting as a sponge for miR-7150. Additionally, IFIT3 expression correlated positively with immune infiltration in LUAD. Circ_BBS9 has been identified as a tumor suppressor in lung adenocarcinoma (LUAD) and holds promise as a diagnostic biomarker. The potential mechanism of action involves the modulation of ferroptosis and the immunological microenvironment through direct interaction with IFIT3 and competitive binding to miR-7150. The aforementioned findings offer new perspectives on the pathophysiology of LUAD and highlight circ_BBS9 as a potentially valuable target for therapeutic interventions.

Effect of CBD on anti-tumor immune response and interaction with GPR55 on MAPK signaling in head and neck squamous cell carcinoma.

6060 Background: Head and neck squamous cell carcinoma (HNSCC) is a lethal malignancy and comprises two distinct etiology: human papillomavirus (HPV)+ve and -ve. Marijuana use is associated with HPV+ oropharyngeal infection and conflicting data show positive and negative associations of daily marijuana use with HPV+ and tobacco-associated HPV- HNSCC. However, cannabinoid use continues to increase in the US general population for recreational purposes as well as in cancer patients for palliative care. G-protein coupled receptors (GPCRs), including cannabinoid GPCRs, stimulated by specific ligands interact with multiple G proteins creating diversity and complexity of signaling. In this study, we aimed to investigate the CBD-induced GPCR coupling and its effect on anti-tumor activity by modulating immune response in HNSCC by using pre-clinical models. Methods: The anti-cancer effect of CBD was measured by cell proliferation, apoptosis and migration analysis. Next, TGF-α shedding assay was performed to identify the CBD-induced GPCR coupling with different cannabinoid receptors. The cannabinoid receptor GPR55 was subjected to silencing by siRNA and western blot analysis was performed to evaluate its role in activation of p38 MPAK pathway. Next, the anti-tumor immune response of CBD was evaluated by using an immunocompetent syngeneic C57BL/6 mEER RasG12D+/- HPV E6/E7 transformed tongue epithelial cell model that serves as a model for HPV+ HNSCC and the 4MOSC1 orthotopic, syngeneic, 4NQO (4-nitroquinoline-1 oxide)-induced murine oral tongue squamous cell carcinoma C57BL/6 mouse model that serve as HPV- HNSCC model as well as in immune-deficient Rag1 -/- and athymic nude mouse followed by measurement of immune cell infiltration by IHC analysis. Results: We found that 10μM of CBD treatment inhibited cell proliferation and migration in HNSCC cells by promoting apoptosis. Interestingly, we observed that CBD resulted in GPCR coupling for Gαi with GPR55 and Gαq/o and Gαq/s for CNR1, CNR2 and GPR55. Furthermore, we silenced GPR55 and observed that 10μM of CBD treatment inhibits the activation of p38 MAPK pathway whereas it promotes activation in non-silenced HNSCC cells. Next, we observed that treatment with 10μM of CBD significantly inhibited tumor growth compared to control in both HPV- and HPV+ models in immune-intact animals whereas, no significant difference in the tumor growth was observed in immune-deficient (Rag1 -/- and athymic nude) mouse, indicating role of CBD in modulating tumor immune microenvironment. We also observed CBD treatment enhanced CD4+ and CD8+ T cell infiltration into the primary tumors of HPV+ and HPV- mice models, implicating a downstream cytotoxic T cell response. Conclusions: Our study suggests that CBD promotes anti-tumor activity by modulating the immune microenvironment and interacts with GPR55 to activate intrinsic MAPK signaling pathway.

Knocking down of Xkr8 enhances chemotherapy efficacy through modulating tumor immune microenvironment

Scramblase Xk-related protein 8 (Xkr8) regulates the externalization of phosphatidylserine (PS) during apoptosis and holds a pivotal role in fostering tumor immunosuppression. Targeting Xkr8 in conjunction with chemotherapy demonstrated a novel avenue for amplifying antitumor immune response and overcoming chemo-immune resistance. Here we further evaluated this strategy by using a clinically relevant orthotopic model and elucidated the mechanism through in-depth single-cell RNA sequencing (scRNA-seq). We found that Xkr8 knockdown exhibited the potential to lead to immunogenic cell death (ICD) by impeding the normal clearance of apoptotic cells. Co-delivery of Xkr8 small interference RNA (siRNA) and a prodrug conjugate of 5-fluorouracil (5-Fu) and oxoplatin (FuOXP) showed remarkable therapeutic efficacy in an orthotopic pancreatic tumor model with increased infiltration of proliferative NK cells and activated macrophages in the tumor microenvironment (TME). Single-cell trajectory analysis further unveiled that tumor infiltrating CD8+ T cells are differentiated favorably to cytotoxic over exhausted phenotype after combination treatment. Our study sheds new light on the impact of Xkr8 knockdown on TME and solidifies the rationale of combining Xkr8 knockdown with chemotherapy to treat various types of cancers.

Identification of two distinct head and neck squamous cell carcinoma subtypes based on fatty acid metabolism-related signatures: Implications for immunotherapy and chemotherapy.

The dysregulation of lipid metabolism is a critical factor in the initiation and progression of tumors. In this investigation, we aim to characterize the molecular subtypes of head and neck squamous cell carcinoma (HNSCC) based on their association with fatty acid metabolism and develop a prognostic risk model. The transcriptomic and clinical data about HNSCC were obtained from public databases. Clustering analysis was conducted on fatty acid metabolism genes (FAMG) associated with prognosis, utilizing the non-negative matrix factorization algorithm. The immune infiltration, response to immune therapy, and drug sensitivity between molecular subtypes were evaluated. Differential expression genes were identified between subtypes, and a prognostic model was constructed using Cox regression analyses. A nomogram for HNSCC was constructed and evaluated. Thirty FAMGs have been found to exhibit differential expression in HNSCC, out of which three are associated with HNSCC prognosis. By performing clustering analysis on these 3 genes, 2 distinct molecular subtypes of HNSCC were identified that exhibit significant heterogeneity in prognosis, immune landscape, and treatment response. Using a set of 7778 genes that displayed differential expression between the 2 molecular subtypes, a prognostic risk model for HNSCC was constructed comprising 11 genes. This model has the ability to stratify HNSCC patients into high-risk and low-risk groups, which exhibit significant differences in prognosis, immune infiltration, and immune therapy response. Moreover, our data suggest that this risk model is negatively correlated with B cells and most T cells, but positively correlated with macrophages, mast cells, and dendritic cells. Ultimately, we constructed a nomogram incorporating both the risk signature and radiotherapy, which has demonstrated exceptional performance in predicting prognosis for HNSCC patients. A molecular classification system and prognostic risk models were developed for HNSCC based on FAMGs. This study revealed the potential involvement of FAMGs in modulating tumor immune microenvironment and response to treatment.

Abstract CT094: Targeting CSF1R with BLZ945 results in effective peripheral and tumor immune microenvironment modulation in advanced solid tumors and may be associated with limited efficacy in recurrent non-mesenchymal glioblastoma

Abstract Colony-stimulating factor 1 receptor (CSF-1R) signaling is involved in regulating monocyte differentiation, and the recruitment and survival of tumor-associated macrophages (TAMs). BLZ945 is a small molecule, highly selective, orally bioavailable, potent kinase inhibitor of CSF-1R that has shown the ability to reduce the recruitment of TAMs in animal models and promote infiltration of effector T cells into the tumor in preclinical models. Given the potential effects of BLZ945 on effector T-cell activity, a combination of BLZ945 with the programmed death 1 (PD-1) inhibitor spartalizumab was investigated in 1) a dose escalation phase I clinical trial with advanced solid tumor patients; and 2) an expansion phase II clinical trial in adult patients with relapsed/refractory glioblastoma (GBM) after standard of care and other lines of therapy (NCT02829723). The percentages of non-classical and intermediate monocytes in the PBMC fraction were decreased after treatment at multiple doses and schedules of treatment tested in the escalation phase. Screening and on-treatment paired tumor biopsy samples from the escalation phase were analyzed by RNA sequencing. Downregulated genes have been noted as macrophage-specific genes, suggesting that treatment with BLZ945 is downregulating macrophages in the tumor microenvironment. There appeared to be a trend for a reduction in TAM gene signatures and increase in T-cell inflammation signatures upon treatment with single-agent BLZ945 or BLZ945 + spartalizumab. RNA-sequencing analysis from archival surgical resection GBM samples from the expansion phase, revealed a potential correlation between PDGFRA gene expression and best percent change in tumor size. This suggests that non-mesenchymal GBM, for which PDGFRA is part of the defining signature, are more sensitive to the treatment. DNA-sequencing of these archival samples also revealed mutations in p53 in the tumors with partial responses but no mutations in EGFR or TERT. Citation Format: Carlos Rodrigo Gil Del Alcazar, Virginia Xu, Chia-chi Lin, Marta Gil-Martin, Aung Naing, Liqiong Fan, Fang Yang, David Quinn, Jincheng Wu, Cornelia Quadt, Jennifer Mataraza. Targeting CSF1R with BLZ945 results in effective peripheral and tumor immune microenvironment modulation in advanced solid tumors and may be associated with limited efficacy in recurrent non-mesenchymal glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT094.

Knocking down of Xkr8 enhances chemotherapy efficacy through modulating tumor immune microenvironment

Scramblase Xkr8 regulates the externalization of phosphatidylserine (PS) during apoptosis and holds a pivotal role in fostering tumor immunosuppression. Targeting Xkr8 in conjunction with chemotherapy demonstrated a novel avenue for amplifying antitumor immune response and overcoming chemo-immune resistance. Here we further evaluated this strategy by using a clinically relevant orthotopic model and elucidated the mechanism through in-depth single-cell RNA sequencing (scRNA-seq). We found that Xkr8 knockdown exhibited the potential to lead to immunogenic cell death (ICD) by impeding the normal clearance of apoptotic cells. Co-delivery of Xkr8 small interference RNA (siRNA) and chemo prodrug FuOXP showed remarkable therapeutic efficacy in an orthotopic pancreatic tumor model with an increase of proliferative NK cells and activated macrophages infiltration in the tumor microenvironment (TME). Single-cell trajectory analysis further unveiled that tumor infiltrating CD8+ T cells are differentiated favorably to cytotoxic over exhausted phenotype after combination treatment. Our study sheds new light on the impact of Xkr8 knockdown on TME and solidifies the rationale of combining Xkr8 knockdown with chemotherapy to treat various types of cancers.

Efficacy of combined treatment with pirfenidone and PD-L1 inhibitor in mice bearing ectopic bladder cancer xenograft

To assess the efficacy of pirfenidone combined with PD-L1 inhibitor for treatment of bladder cancer in a mouse model and its effect on tumor immune microenvironment modulation. Forty C57BL/6 mouse models bearing ectopic human bladder cancer xenografts were randomized into control group, PD-L1 inhibitor group, pirfenidone group and combined treatment group (n=10). After successful modeling, PD-L1 inhibitor treatment was administered via intraperitoneal injection at 12.5 mg/kg every 3 days, and oral pirfenidone (500 mg/kg) was given on a daily basis. The survival rate of the mice and tumor growth rate were compared among the 4 groups. The expressions of CD3, CD8, CD45, E-cadherin and N-cadherin in the tumor tissues were detected with immunohistochemistry after the 21-day treatment, and bone marrow-derived suppressor cells (MDSCs) were observed with immunofluorescence staining; serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), creatinine (CRE) and lactate dehydrogenase (LDH-L) were analyzed using an automated biochemical analyzer. Treatment with PD-L1 inhibitor and pirfenidone alone both significantly decreased tumor growth rate and tumor volume at 21 days (P < 0.05), but the combined treatment produced an obviously stronger inhibitory effect (P < 0.05). PD-L1 inhibitor and pirfenidone alone significantly increased E- cadherin expression and decreased N-cadherin expression in the tumor tissue (P < 0.05). The two treatments both significantly increased the percentage of CD3+, CD8 and CD45+ T cells and decreased the percentage of Ly-6G+CD11b+MDSCs in the tumor tissue, and these changes were more obvious in the combined treatment group (P < 0.05). No significant differences were found in serum ALT, AST, BUN, CRE or LDH-L levels among the 4 groups (P>0.05). Combined treatment with pirfenidone and PD-L1 inhibitor significantly inhibits the progression of bladder cancer in mice possibly by regulating tumor immune microenvironment and inhibiting epithelial-mesenchymal transition of the tumor cells.

Surufatinib plus sintilimab in patients with advanced gastric/gastroesophageal junction adenocarcinoma (GC/GEJC): A single-arm, open-label, single-center phase II trial.

358 Background: Anti-PD-1 antibodies plus chemotherapy have become the standard first-line treatment for advanced GC/GEJC with CPS score ≥ 5. However, GC/GEJC still has dismal survival. Preclinical studies have demonstrated that surufatinib (a novel small-molecule kinase inhibitor targeting VEGFR1-3, FGFR and CSF-1R) combined with anti-PD-1 antibodies have synergistic antitumor effects by modulating tumor immune microenvironment. Therefore, a phase II trial to evaluate surufatinib plus sintilimab in patients (pts) with advanced GC/GEJC was conducted. Here we report the results of part A of this trial. Methods: This single arm, open-label, single center phase II trial consisted of two parts: part A (second-line therapy) and part B (first-line therapy). In part A, pts aged 18-75 years who were HER2-negative, failed first-line standard therapy, and immune checkpoint inhibitor therapy naive were enrolled. Part A included safety run-in and dose expansion stages. Six pts in the safety run-in received surufatinib at 250mg once daily as starting dose, in combination with a fixed dose of sintilimab (200 mg, d1, Q3W). If more than 1 DLT occurred in stage 1, the recommended dose of surufatinib for stage 2 (N=11) would de-escalate to 200 mg, otherwise would maintain as 250mg. The primary endpoint was ORR per RECIST v1.1. Secondary endpoints included DCR, PFS, OS and safety. If the ORR of part A was ≥ 30%, the trial would proceed to Part B (N=44), further evaluating this combination treatment in previously untreated advanced GC/GEJC. Results: At data cutoff on 8/10/2023, 16 pts (6 in safety run-in; 10 in dose expansion) were enrolled in part A. The median age was 56 years; 75% male; 69% with ECOG PS 1. Peritoneum (56%) was the most common metastatic site. No DLTs were observed in the safety run-in stage, so the recommended dose of surufatinib for dose expanding stage was 250 mg. Among the 16 pts evaluable for tumor response, 6 pts achieved PR, 4 pts achieved SD. The confirmed ORR and DCR per RECIST v1.1 was 40.0% (95% CI: 15.2- 64.6%) and 66.7% (95% CI: 38.4- 88.2%), respectively. Median DOR was not reached and 50% of responders were estimated to have a response duration ≥ 7.8 months. Median PFS and OS were not yet mature. Most TEAEs (≥ 40%) were grade 1-2, including hypoproteinemia (62.5%), hypertension (50%), proteinuria (50%), lymphocyte count decreased (43.8%). The most common grade ≥ 3 TEAEs reported by 5 pts (31.3%) were blood bilirubin increased (18.8%), hypertension, lymphocyte count decreased, and alanine transaminase elevation (6.3%, respectively). No treatment-related death was reported. Conclusions: The results showed surufatinib plus sintilimab acquired preliminary efficacy and manageable safety profile as the second-line treatment for advanced GC/GEJC. Part B is ongoing, and more data analysis will be reported further. Clinical trial information: NCT05235906 .

FBLN5 as One Presumably Prognostic Gene Potentially Modulating Tumor Immune Microenvironment for Renal Clear Cell Carcinoma in Children and Young Adults.

To investigate the role of FBLN5in renal clear cell carcinoma (KIRC), in particular on the tumor's immune microenvironment, including children and young adults. FBLN5 expression in tumor and normal samples was explored using SangerBox, TIMER2.0, GEPIA, UALCAN, HPA databases. The Linkedomics database was used to obtain FBLN5 co-expressed genes in KIRC tissue. SangerBox was also used to estimate immune infiltration of FBLN5 in KIRC. The Kaplan-Meier plotter was used to investigate the survival effects of FBLN5 expression in the presence of immune infiltration. We then collected 48 cases from 7 hospitals over a-20 year period to calculate the impact of FBLN5 on the prognosis of children and young adults with KIRC. FBLN5 expression was significantly reduced in KIRC tissue compared to normal adjacent tissue. FBLN5 was potentially involved in the immune-related biological processes. In addition, FBLN5 expression has been linked to a number of immune checkpoints, cytokines, chemokines and chemokine receptors in KIRC. At the same time, the expression of FBLN5 affected the survival rates differently in KIRC patients with high or low levels of immune infiltration. High expression of FBLN5 in children and young adults with KIRC was associated with a favorable prognosis. This study shed light on the potential of FBLN5 as a prognostic marker in children and young adults with KIRC and as an immune-related target for clinical treatment.

Prognostic, immunity, stemness, and anticancer drug sensitivity characterization of pyroptosis related genes in non-small cell lung cancer.

Pyroptosis plays a pivotal role in the tumor immune microenvironment (TME) dynamics, particularly in non-small cell lung cancer (NSCLC). The aim of our study was to explore its effects on tumor progression, TME patterns, and the efficacy of therapeutic interventions in NSCLC. Our investigation encompassed a thorough analysis of pyroptosis-related genes (PRGs), integrating immunohistochemistry (IHC) data, TME characteristics, stemness indices, and anticancer drug sensitivities. We aimed to analyze mRNA expression profiles across various cancers, constructing benchmark datasets to assess the clinical significance of PRGs in NSCLC. This included evaluating their association with clinical responses and efficacy. Notably, both our and HPA IHC data demonstrated significantly elevated GSDMD-N protein levels in lung squamous cell carcinoma (LUSC) tissues. The expression of PRGs differed significantly between tumor and normal tissues across various cancers, as validated by IHC data, and was correlated with prognosis (p < 0.05). Moreover, our investigation revealed significant differences (p < 0.05) in the expression of the PRGs among distinct TME subtypes categorized as C1 (wound healing), C3 (inflammatory), C2 (IFN-gamma dominant), C5 (immunological quiet), C4 (lymphocyte deficient), and C6 (TGF-beta dominant). Additionally, our research on anticancer drug sensitivity uncovered compelling connections between specific anticancer medications and the expression of PRGs, including GSDMD, ELANE, IL18, and CHMP4A (p < 0.05). Our study provided valuable insights into the critical role of PRGs in TME modulation, tumor stemness, and anticancer drug sensitivity across diverse cancers. Our findings illuminate the intricate relationship between pyroptosis and the TME, offering new perspectives for enhancing NSCLC treatment and prognosis.

Immunostimulatory gene therapy combined with checkpoint blockade reshapes tumor microenvironment and enhances ovarian cancer immunotherapy

Immune evasion has made ovarian cancer notorious for its refractory features, making the development of immunotherapy highly appealing to ovarian cancer treatment. The immune-stimulating cytokine IL-12 exhibits excellent antitumor activities. However, IL-12 can induce IFN-γ release and subsequently upregulate PDL-1 expression on tumor cells. Therefore, the tumor-targeting folate-modified delivery system F-DPC is constructed for concurrent delivery of IL-12 encoding gene and small molecular PDL-1 inhibitor (iPDL-1) to reduce immune escape and boost anti-tumor immunity. The physicochemical characteristics, gene transfection efficiency of the F-DPC nanoparticles in ovarian cancer cells are analyzed. The immune-modulation effects of combination therapy on different immune cells are also studied. Results show that compared with non-folate-modified vector, folate-modified F-DPC can improve the targeting of ovarian cancer and enhance the transfection efficiency of pIL-12. The underlying anti-tumor mechanisms include the regulation of T cells proliferation and activation, NK activation, macrophage polarization and DC maturation. The F-DPC/pIL-12/iPDL-1 complexes have shown outstanding antitumor effects and low toxicity in peritoneal model of ovarian cancer in mice. Taken together, our work provides new insights into ovarian cancer immunotherapy. Novel F-DPC/pIL-12/iPDL-1 complexes are revealed to exert prominent anti-tumor effect by modulating tumor immune microenvironment and preventing immune escape and might be a promising treatment option for ovarian cancer treatment.

A phase Ib/II study to evaluate surufatinib combined with camrelizumab and chemotherapy in the second-line treatment of advanced colorectal cancer: Phase Ib results.

3555 Background: Surufatinib is a novel small-molecule kinase inhibitor targeting VEGFR1-3, FGFR and CSF-1R with confirmed efficacy in epNET and pNET. Preclinical studies have demonstrated that surufatinib combined with immune checkpoint inhibitors (ICIs) have synergistic antitumor effects by modulating tumor immune microenvironment. Additionally, surufatinib plus toripalimab (anti-PD-1) has showed preliminary anti-tumor activity in multiple solid tumors, including colorectal cancer (CRC). This study aimed to evaluate the efficacy and safety of surufatinib plus camrelizumab (anti-PD-1), irinotecan and GM-CSF as second-line treatment in advanced CRC. Here we reported the results of the phase Ib study. Methods: This ongoing phase Ib/II, open-label trial (NCT04929652) enrolled patients (pts) aged 18-75 years old with pathologically confirmed locally advanced or metastatic CRC who have progressed on/been intolerant to the standard first-line therapy. In the Ib dose escalation/de-escalation phase, pts received surufatinib at 250mg once daily (QD) as starting dose, in combination with fixed dose of camrelizumab (200 mg, d1), irinotecan (200 mg/m2, d1) and GM-CSF (5ug/kg, d2-d7), every 3 weeks (Q3W). Based on DLTs, the dose of surufatinib should escalate to 300 mg or de-escalate to 200 mg. The primary objective of the phase Ib study was to determine the safety and DLT in first treatment cycle defining the recommended phase 2 dose (PR2D). Additional 36 pts were enrolled in the phase II dose expansion stage using RP2D. Primary endpoint of phase II was ORR as per RECIST 1.1. Secondary endpoints included PFS, DCR, OS and safety. Results: Enrollment opened in Nov 2021 and data cutoff in Dec 2022. 12 pts (median age 54) were treated in Phase Ib study (surufatinib 250 mg: N = 6; surufatinib 300 mg: N = 6). 75% were male and 17% had ECOG PS 1. 11/12 (92%) pts were positive for KRAS, NRAS or BRAF mutations. No DLT was observed in the starting dose cohort (surufatinib 250 mg), and one patient in the surufatinib 300 mg cohort experienced DLT (Grade [G] 3 vomiting). Surufatinib 300 mg QD was defined as the RP2D. Other G3 treatment related adverse events (TRAEs) were hypertension (1/12; 8.3%) and diarrhea (1/12; 8.3%). All other TRAEs were grade 1-2, and the most common (≥40%) were diarrhea (75%), fatigue (58%), vomiting (58%), nausea (50%), hypertension (42%), proteinuria (42%) and alopecia (42%). Of the 12 pts evaluable for tumor response, 3 pts achieved PR, 9 pts achieved SD. The ORR was 25% (3/12), the DCR was 100% (12/12) and median PFS was 7.2 months (95%CI 3.7-10.7). Conclusions: Surufatinib plus camrelizumab, irinotecan and GM-CSF was well tolerated, with a manageable safety profile, and showed preliminary anti-tumor activity in patients with advanced CRC. The dose-expansion phase is ongoing. Clinical trial information: NCT04929652 .

Effect of locoregional injection of RNA oligonucleotide targeting carbohydrate sulfotransferase 15 on both tumoral and lymph node MDSCs and T cell infiltration in pancreatic tumor in mice.

e16310 Background: Carbohydrate sulfotransferase 15 (CHST15) is a proteoglycan biosynthetic enzyme and is responsible for tumor matrix remodeling. Intratumoral injection of CHST15 siRNA was shown to increase tumor-infiltrating T cells in patients with pancreatic cancer in the phase I/IIa study. However, the mechanism underlying the enhanced T cell accumulation is not fully explored. Here we investigated the effect of locally injected CHST15 siRNA on modulating tumor immune microenvironment in mice. Methods: For in vivo pharmacokinetics after a single local injection of CHST15 siRNA to normal C57BL/6J mice, RNA oligonucleotides were isolated from tissues and detected by HPLC. Mouse pancreatic cancer KPC cells were implanted subcutaneously into the left hind footpad of syngeneic C57BL/6J mice and the tumor growth was monitored. From week 1, intratumoral injections with 1 mg/mL of CHST15 siRNA or vehicle were performed twice a week for 2 weeks and mice were sacrificed at week 3. In separate experiments, human BxPC-3 cells were implanted subcutaneously in T cell-deficient nude mice, and the effect of intratumoral injections of CHST15 siRNA or vehicle for 2 weeks was also investigated. Immunohistochemical staining was performed for the tumor and tumor-draining lymph node (TDLN). Results: CHST15 siRNA was detectable in not only the local sites but also the draining LNs of normal mice 1 and 4 hours after locoregional injection. In the KPC-implanted syngeneic mouse model, CHST15 siRNA showed significant tumor growth inhibition compared to the vehicle. In tumor, CHST15 was expressed by tumor cells, and to a lesser extent, Ly6C/G+ MDSCs. CHST15 siRNA altered tissue remodeling by repressing dense fibrotic architecture and NETosis. CHST15 siRNA significantly diminished Ly6C/G+ MDSCs while increasing CD4+ and CD8+ T cells. In TDLN, CHST15 was expressed by Ly6C/G+ MDSCs. Locally injected CHST15 siRNA markedly diminished Ly6C/G+ MDSCs, while increasing Ki-67+ proliferating CD4+ and CD8+ T cells. In the BxPC-3-implanted nude mouse model, locally injected CHST15 siRNA repressed excessive fibrosis and NETosis in tumor, diminishing both tumoral and TDLN MDSCs, but tumor growth inhibition was marginal. These results suggest that locally injected CHST15 siRNA could enter into TDLN, directly diminishing CHST15+ MDSCs, and expanded T cells. Tumor growth inhibition was largely T-cell medicated. Conclusions: The present study demonstrated that CHST15 siRNA locally and remotely diminished MDSCs, leading to the enhancement of anti-tumor T cell activity. A novel role of locally-delivered RNA oligonucleotide is suggested.

Modulation of tumor immune microenvironment by TAS-115, a multi-receptor tyrosine kinase inhibitor, promotes antitumor immunity and contributes anti-PD-1 antibody therapy

TAS-115 is an oral multi-receptor tyrosine kinase inhibitor that strongly inhibits kinases implicated in antitumor immunity, such as colony stimulating factor 1 receptor and vascular endothelial growth factor receptor. Because these kinases are associated with the modulation of immune pathways, we investigated the immunomodulatory activity of TAS-115. An in vitro cytokine assay revealed that TAS-115 upregulated interferon γ (IFNγ) and interleukin-2 secretion by T cells, suggesting that TAS-115 activated T cells. Gene expression analysis suggested that TAS-115 promoted M1 macrophage differentiation. In in vivo experiments, although TAS-115 exerted a moderate antitumor effect in the MC38 mouse colorectal cancer model under immunodeficient conditions, this effect was enhanced under immunocompetent conditions. Furthermore, combination of TAS-115 and anti-PD-1 antibody exhibited greater antitumor activity than either treatment alone. Flow cytometry analysis showed the increase in IFNγ- and granzyme B (Gzmb)-secreting tumor-infiltrating T cells by TAS-115 treatment. The combination treatment further increased the percentage of Gzmb+CD8+ T cells and decreased the percentage of macrophages compared with either treatment alone. These results highlight the potential therapeutic effect of TAS-115 in combination with PD-1 blockade, mediated via activation of antitumor immunity by TAS-115.

Integrated analysis of single-cell RNA-seq and bulk RNA-seq unravels T cell-related prognostic risk model and tumor immune microenvironment modulation in triple-negative breast cancer

Triple negative breast cancer (TNBC) is an aggressive and fatal malignancy. The current success of tumor immunotherapy has focused attention on intermediate T-cell subsets and the tumor microenvironment, which are essential for activation of the anti-tumor response. Therefore, both areas require further research to accelerate progress in developing tailored immunotherapeutic approaches for patients with TNBC. We obtained scRNA-seq data of TNBC from the GEO database. A multiplex strategy was used to analyze and identify the T-cell heterogeneity of TNBC. By combining the METABRIC and GEO databases, a prognostic risk model for T-cell marker genes was constructed and validated. In addition, the immune-infiltrating cells of TNBC was analyzed using CIBERSORT, and the association between the risk model and response to immunotherapy was investigated. Based on scRNA-seq data, 25,932cells were identified for multiple analyzes. T cells were studied with a focus on 2 subtypes, including CD8+ and CD4+. There were also communication relationships between T cells and multiple cell types. The results of the enrichment analysis showed that the T-cell marker genes were focused in pathways related to the immune system. In addition, OPTN, TMEM176A, PKM and HES1 deserve attention as prognostic markers in TNBC. The immune infiltration results showed that the high-risk group had significant immune cell infiltration and immunosuppression status. This study provides a resource for understanding T-cell heterogeneity and the associated prognostic risk model for TNBC. The results show that the model helps predict prognosis and response to treatment in breast cancer.

Abstract 6677: Preliminary immune correlatives from BCA101 trial show favorable modulation of tumor immune microenvironment

Abstract Background: BCA101 is a bispecific antibody targeting EGFR and TGF-β. TGF-β pathway activation is a hallmark of human immune-excluded tumors, and TGF-β expression is associated with resistance to anti-PD-1 blockade. Neutralization of TGF-β removes an immunosuppressive signal that drives accumulation and polarization of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in solid tumors, while EGFR inhibition targets tumor cell-intrinsic oncogenic signaling. Co-targeting of EGFR and TGF-β directly impacts tumor progression while enhancing the immunogenicity of tumors. Methods: Patients with multiple solid tumor types (CRC, pancreatic, HNSCC, SqNSCLC, and others) were treated with escalating doses of either single agent BCA101 or in combination with anti-PD-1 (pembrolizumab) enrolled on NCT04429542 trial. We performed a variety of immune correlatives on pre- and on-treatment tumor biopsies, including Nanostring-based transcriptomic profiling and IHC for immunophenotypic markers, as well as multiparametric flow cytometric profiling of circulating PBMCs. Results: Our preliminary evidence suggests that neutralization of TGF-β positively alters the systemic immune state (PBMCs) and tumor immune phenotype (mRNA, IHC). Circulating HLA-DR+ monocytes were significantly increased in on-treatment PBMC samples relative to screening. Pathway analysis of on-treatment tumor biopsies revealed enhanced costimulatory signaling, cytokine and chemokine signaling, immune infiltration, and interferon signaling. Top differentially regulated genes in on-treatment biopsies included CCL21, CXCL9, CXCL11, and CXCL13, which recruit T and NK cells. HDAC11, which negative regulates type-I interferon signaling, was significantly reduced in on-treatment biopsies. Notably, two patients with EGFR-amplified squamous non-small cell lung cancer, who both progressed on first-line immunotherapy treatment, were treated with BCA101 at 1250 mg and 1500 mg qw and achieved a partial response (ongoing for 10 months at the time of the data cutoff) and a prolonged stable disease for 11 months, respectively. They exhibited increased CD8+ T cell infiltration and a reduction in TAMs following treatment. Conclusions: Increased abundance of circulating HLA-DR+ monocytes following treatment indicated polarization towards a more positive, Th1-like systemic immune state. We observed enhanced immunogenicity of tumors as assessed by a targeted IO transcriptomic analysis. The results of the pathway analysis were supported by IHC on post-treatment biopsies from a subsequent cohort showing enhanced CD8+ T cell infiltration and stable, or reduced expression of TAM marker CD163. These results indicate that neutralization of TGF-β induces a more permissive tumor immune microenvironment. Citation Format: Patrick H. Lizotte, Paul Paik, Liviu Niculescu, Seng-Lai H. Tan, David Bohr, Elham Gharakhani, Ralf Reiners, Rachel Salazar, Avanish Varshney, Shiv Ram Krishn, Pradip Nair, Cloud Paweletz. Preliminary immune correlatives from BCA101 trial show favorable modulation of tumor immune 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 6677.

Abstract 638: EPB-53 prevents NASH-HCC transition via regulation of SPHK1-S1P-HIPPO signaling and immune modulation in a murine model

Abstract Background: Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of disorders, ranging from non-progressive bland steatosis to non-alcoholic hepatitis (NASH), cirrhosis, and malignant transformation into hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) exerts a key role in lipid homeostasis and play a promising therapeutic candidate for the treatment of obesity, diabetes, and NASH. Small molecules like EPB-53 that are activators of heme-regulated inhibitor (HRI) of eukaryotic initiation factor 2α (eIF2α) kinase have been synthesized to induce FGF21. Previous study has shown that oral administration of EPB-53 alleviates NASH through increased FGF21 to prevent hepatic steatosis. However, it is unknown whether EPB-53 can prevent NASH-HCC transition. Aim: To investigate the effect of EPB-53 on NASH-HCC transition and the therapeutic mechanism, focusing on the lipid metabolic reprogramming and tumor immune microenvironment (TIM). Methods: NASH-HCC transition model was established in two-week-old male FGF21 knockout (KO) mice and wild type controls (C57BL/6J mice) by diethylnitrosamine (DEN) administration at 40 mg/kg body weight (i.p.). Four weeks after DEN administration, the mice were fed with western-style high fat diet (WSHFD, 60% kcal% fat and 19% kcal% fructose) for 3 months, and gavaged daily with either vehicle, 2% w/v, (2-hydroxypropyl)-β-cyclodextrin or EPB-53 (300 mg/kg/day) in vehicle for 4 weeks before sacrifice. The tumor incidence, lipid metabolism, and carcinogenesis signaling pathways were determined. Mass cytometry (CyTOF) analysis was performed to detect the cell subpopulations in the NASH microenvironment and NASH-HCC TIM. Results: Severe NASH with tumor cell detection, aberrant lipid components and HIPPO-YAP signaling were found in liver tissues of FGF21-KO mice compared to the WT controls. The upregulated sphingosine Kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) in liver subsequently induced YAP dephosphorylation and nuclear localization. EPB-53 treatment prevents NASH-HCC transition evident as decreased HCC incidence in WT mice but not in FGFKO mice. Continuously increased FGF21 production in WT mice contributes to down-regulation of SPHK1-S1P-HIPPO-YAP signaling. Interestingly, EPB-53 treatment can alleviate the compromised function of macrophages/Kupffer cells which render a TIM in the NASH-HCC mice. Conclusion: 1) Lack of FGF21 played a key role in lipid metabolic disorder and SPHK1-S1P-HIPPO signaling to promote NASH-HCC transition; 2) Lack of FGF21 rendered a TIM in NASH liver contributing to the NASH-HCC transition; 3) EPB-53 treatment can not only alleviate NASH, but also prevent NASH-HCC transition via lipid metabolic reprogramming, down-regulation of the SPHK1-S1P-HIPPO axis, and modulation of TIM. Citation Format: Min Tan, Eugenia Pujol, Santiago Vazquez, Jingwen Zhang, Jun Yan, Robert C. Martin, Manuel Vazquez-Carrera, Yan Li. EPB-53 prevents NASH-HCC transition via regulation of SPHK1-S1P-HIPPO signaling and immune modulation in a murine 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 638.

Targeted delivery of pexidartinib to tumor-associated macrophages via legumain-sensitive dual-coating nanoparticles for cancer immunotherapy

Tumor-associated macrophage (TAM) is regarded as an appealing cell target for cancer immunotherapy. However, it remains challenging to selectively eliminate M2-like TAM in tumor microenvironment. In this work, we employed a legumain-sensitive dual-coating nanosystem (s-Tpep-NPs) to deliver CSF-1R inhibitor pexidartinib (PLX3397) for targeting TAM therapy. The PLX3397-loaded NPs exhibited uniform size of ∼240 nm in diameter, good drug loading capacity and efficiency, as well as sustained drug release profile. Compared to non-sensitive counterpart ns-Tpep-NPs, s-Tpep-NPs showed distinguished selectivity upon M1 and M2 macrophage uptake with relation to incubation time and dose. Besides, the selectivity of anti-proliferation effect was also identified for s-Tpep-NPs against M1 and M2 macrophage. In vivo imaging demonstrated that s-Tpep-NPs exhibited much higher tumoral accumulation and TAM recognition specificity as compared to non-sensitive ns-Tpep-NPs. In vivo efficacy verified that s-Tpep-NPs formulation was much more effective than ns-Tpep-NPs and other PLX3397 formulations to treat B16F10 melanoma via targeting TAM depletion and modulating tumor immune microenvironment. Overall, this study provides a robust and promising nanomedicine strategy for TAM-targeted cancer immunotherapy.

Abstract OT1-05-02: Efficacy and safety of sintilimab in combination with anlotinib plus metronomic chemotherapy in advanced triple negative breast cancer (SPACE): preliminary results of a single-arm, multicenter phase II trial

Abstract Background: Anti-PD-1 antibody combined with antiangiogenic drugs have demonstrated antitumor activity in advanced triple negative breast cancer (TNBC). Anlotinib, an oral multi-targeted tyrosine kinase inhibitor (TKI), has synergistic effect with anti-PD-1 antibody. Preclinical studies showed that metronomic chemotherapy inhibited angiogenesis and enhanced immunotherapy efficacy in TNBC via modulation of tumor immune microenvironment. We hereby conducted a single-arm, multicenter, phase II trial to investigate the efficacy and safety of sintilimab (anti-PD-1 antibody) in combination with anlotinib plus metronomic chemotherapy as a potential novel therapeutic strategy in advanced TNBC and explore potential biomarkers. Methods: Forty-three cases were planning to be included in this trial. The eligible patients who had received no more than two lines of chemotherapy for metastatic disease were enrolled and received sintilimab (200 mg iv q3w) in combination with anlotinib (12 mg po d1-14 q3w) plus capecitabine (500 mg po, tid) or vinorelbine (40 mg po, tiw) until disease progression or intolerable toxicity. The primary endpoint is objective response rate (ORR) and secondary endpoints are disease control rate (DCR), progression free survival (PFS), and overall survival (OS). The safety profile has also been assessed. Blood samples collected at different time points of the baseline, first and second cycle post-treatment, and disease progression were used for next-generation sequencing of ctDNA containing 654 tumor-related genes. Results: As of July 2022, a total of 32 patients were enrolled, and 29 patients were evaluable for efficacy. 2 patients (6.9%) achieved complete response (CR). 6 patients (20.7%) achieved partial response (PR). The ORR is 27.6% (8/29) and DCR is 79.3% (23/29). The median PFS was not reached. The most common grade 1 or 2 adverse events (AEs) include elevated thyroid stimulating hormone (37.0%, 10/27), hand-foot syndrome (18.5%, 5/27), elevated aspartate aminotransferase (14.8%, 4/27), elevated bilirubin (11.1%, 3/27) and hypertension (11.1%, 3/27). Grade 3 AEs include elevated bilirubin (3.7%, 1/27) and hypertension (3.7%, 1/27). No grade 4 or 5 AEs occurred. By analyzing ctDNA mutations of blood samples in 10 patients at baseline, we found that genes with high mutation frequency were HLA-DRB5 (8/10, 80%), TP53 (7/10, 70%), HLA-DRB1 (5/10, 50%) and PIK3CA (4/10, 40%). Among these 10 patients, 2, 3 and 5 patients achieved PR, SD and PD, respectively. The number of gene mutations in patients with PD was higher than that in patients with PR or SD at baseline. This indicate that mutations in ctDNA may be associated with poor efficacy in advanced TNBC. But this still needs further verification. Dynamic analysis of gene mutations at different time points showed that the amplification of HLA-DRB5 or the elimination of KMT2D, RELN and TP53 occurred in patients with PR and SD, but not in patients with PD. Conclusions: Sintilimab in combination with anlotinib plus metronomic chemotherapy has shown favorable efficacy and acceptable safety profile in patients with advanced TNBC. The clinical significance of ctDNA dynamic monitoring needs further validation. Clinical trial information: ChiCTR2100044725 Citation Format: Huihui Li, Qiaorui Tan, Shujuan Sun, Dongdong Zhou, Bo Yu, Mu Su, Baojiang Li, Shu Fang, Ling Qiang, Guohua Ren, Bing Bu, Sha Yin, Xiaochu Man, Pengfei Qiu, Xinzhao Wang, Chao Li, Fangli Cao, Qian Shao, Dali Han, Lihua Song, Bingjie Fan, Baoxuan Zhang, Liang Xu, Xianguang Zhao, Yuqian Liao, Xuemei Xie, Lanping Liu. Efficacy and safety of sintilimab in combination with anlotinib plus metronomic chemotherapy in advanced triple negative breast cancer (SPACE): preliminary results of a single-arm, multicenter phase II trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-05-02.