Modulation Of Tumor Microenvironment Research Articles

Long non-coding RNAs modulate tumor microenvironment to promote metastasis: novel avenue for therapeutic intervention.

Cancer is a devastating disease and the primary cause of morbidity and mortality worldwide, with cancer metastasis responsible for 90% of cancer-related deaths. Cancer metastasis is a multistep process characterized by spreading of cancer cells from the primary tumor and acquiring molecular and phenotypic changes that enable them to expand and colonize in distant organs. Despite recent advancements, the underlying molecular mechanism(s) of cancer metastasis is limited and requires further exploration. In addition to genetic alterations, epigenetic changes have been demonstrated to play an important role in the development of cancer metastasis. Long non-coding RNAs (lncRNAs) are considered one of the most critical epigenetic regulators. By regulating signaling pathways and acting as decoys, guides, and scaffolds, they modulate key molecules in every step of cancer metastasis such as dissemination of carcinoma cells, intravascular transit, and metastatic colonization. Gaining a good knowledge of the detailed molecular basis underlying lncRNAs regulating cancer metastasis may provide previously unknown therapeutic and diagnostic lncRNAs for patients with metastatic disease. In this review, we concentrate on the molecular mechanisms underlying lncRNAs in the regulation of cancer metastasis, the cross-talk with metabolic reprogramming, modulating cancer cell anoikis resistance, influencing metastatic microenvironment, and the interaction with pre-metastatic niche formation. In addition, we also discuss the clinical utility and therapeutic potential of lncRNAs for cancer treatment. Finally, we also represent areas for future research in this rapidly developing field.

Abstract IA019: Tumor microenvironment modulation is critical for cellular immunotherapy efficacy

Abstract Background: Chimeric antigen receptor (CAR)-engineered T cell therapies are being pursued for the treatment of prostate cancer, among other solid tumors. However the role of lymphodepletion was uncertain for application outside of heme malignancies. We studied the effect of chemotherapy preconditioning on the tumor immune microenvironment along with the impact on CAR-T cell activity. Methods: Immunocompetent mice bearing RM9 prostate tumors lentivirally transduced to express human prostate stem cell antigen (PSCA) and treated with PSCA targeted CAR T cells alone, or after 100 mg/kg cyclophosphamide (Cy). Tumors were evaluated for immune cell subsets infiltration in addition to clinical response. As part of a phase 1 dose escalation trial patients received 100 M PSCA-targeted CAR T cells, manufactured at City of Hope’s cGMP facility, alone or after Cy. CAR T persistence and cytokine levels were evaluated by flow cytometry and luminex, respectively. Results: CAR T cells alone had minimal anti-tumor effect in immune competent mice. Cy was associated with increased CD11c+ DCs and reduced CD206+ M2 macrophages in the tumors. With Cy pre-conditioning followed by infusion of PSCA-CAR T cells, over 40% of mice had complete response (CR) and rejected rechallenge of tumor. 10-fold greater T cell infiltration was noted in tumors after Cy preconditioning, with lesser increase and no anti-tumor effect noted with Cy + mock CAR T cells. In the phase 1 human trial greater CAR T cell expansion and cytokine induction were noted with Cy + PSCA-CAR T compared to PSCA-CAR T alone. Conclusion: Cy functions as a modifier of the prostate cancer tumor microenvironment, facilitating CAR T cell infiltration and expansion. This has resulted in greater activity of PSCA-CAR T cells both in murine models and preliminary human testing. Further study of manipulations to optimize the tumor immune microenvironment will likely be important to maximizing efficacy of cellular immunotherapy in prostate cancer. Citation Format: Tanya B Dorff, M Suzette Blanchard, John Murad, Lauren Adkins, Gaurav Dhaploa, Stephen Forman, Saul Priceman. Tumor microenvironment modulation is critical for cellular immunotherapy efficacy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr IA019.

Light-responsive nanomedicine for cancer immunotherapy.

Immunotherapy emerged as a paradigm shift in cancer treatments, which can effectively inhibit cancer progression by activating the immune system. Remarkable clinical outcomes have been achieved through recent advances in cancer immunotherapy, including checkpoint blockades, adoptive cellular therapy, cancer vaccine, and tumor microenvironment modulation. However, extending the application of immunotherapy in cancer patients has been limited by the low response rate and side effects such as autoimmune toxicities. With great progress being made in nanotechnology, nanomedicine has been exploited to overcome biological barriers for drug delivery. Given the spatiotemporal control, light-responsive nanomedicine is of great interest in designing precise modality for cancer immunotherapy. Herein, we summarized current research utilizing light-responsive nanoplatforms to enhance checkpoint blockade immunotherapy, facilitate targeted delivery of cancer vaccines, activate immune cell functions, and modulate tumor microenvironment. The clinical translation potential of those designs is highlighted and challenges for the next breakthrough in cancer immunotherapy are discussed.

Characterization of MCL-1 in patients with colorectal cancer (CRC): Expression, molecular profiles, and outcomes.

3085 Background: Myeloid cell leukemia 1 (MCL-1) is a member of the BCL-2 protein family and is anti-apoptotic/pro-survival in function. Dysregulation of MCL-1 expression has been reported in several solid tumors, including lung and breast cancer. In CRC, MCL-1 has been associated with resistance to chemotherapeutic drugs and multi-kinase inhibitor regorafenib. Our study aimed to characterize the molecular features associated with MCL-1 gene expression in CRC. Methods: 28,576 CRC samples were analyzed by Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES). MCL-1 expression was stratified by quartiles where top quartile transcripts per million (TPM) were considered high (Q4) and bottom quartile low (Q1). Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by RNA deconvolution analysis using QuantiSEQ. Interferon-gamma and T-cell inflamed signatures were also calculated from RNA data. X2 and Fisher-Exact tests were used, and statistical significance was determined as a P-value adjusted for multiple comparisons ( q < 0.05). Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results: MCL-1 expression was significantly increased in rectal tumors compared to other primary tumor sites (median TPM [mTPM] 20.8 vs 18.9 vs 18.7, in rectal vs left-sided vs right-sided, respectively, q < 0.0001). MCL-1 high tumors were associated with increased rate of PD-L1 IHC levels (Q1 vs Q4: 4.2% vs 9.7% [all CRC]; 3.7% vs 8.9% [pMMR/MSS], q < 0.0001), while MCL-1-low showed marginally increased rates of TMB-high (8.0% vs 7.0% [all CRC]; 3.3% vs 2.6% [pMMR/MSS], P < 0.05). MCL-1-high expression was associated with a higher T-cell inflamed signature and IFN score ( q < 0.0001). MCL-1 high was associated with higher mutation rates of CDKN2A, BRCA2, KRAS and GNAS, while lower mutation rates of TP53, PIK3CA, PTEN, BRAF, APC, FBXW7, AMER1, SOX9, and SMAD2 and copy number amplifications in several genes ( q < 0.05). MCL-1 high TME had higher CI of M1 and M2 macrophages, monocytes, B cells, NK cells, and T-reg infiltration, while dendritic cells and CD4+ T-cell were lower ( q < 0.05). Patients with high MCL-1 expressing CRC had longer survival (Q4 vs Q1: 33.5 vs 24.8 months, HR 0.72, 95% CI 0.67-0.77, P < 0.0001) and longer time-on-treatment with regorafenib although with limited clinical benefit (Q4 vs Q1: 2.03 vs 1.06 months, HR: 0.67, 95% CI: 0.50-0.89, P = 0.005). Conclusions: Our data show a strong correlation between distinct immune biomarkers, TME cell infiltration and MCL-1 expression in CRC. Furthermore, increased tumor MCL-1 expression improved patient prognosis and treatment outcomes. These findings suggest a key clinical role for MCL-1 as an important modulator of anti-tumor immunity and TME and a potential biomarker in CRC.

Effect of FAPα-expressing tumor cell vaccine with PD-1 mAb on its efficacy in mice colorectal cancer model.

e14615 Background: Colorectal cancer (CRC) is the third prevalent malignant tumor all over the world, and many cases are metastatic at diagnosis. Immunotherapy emerges a promising treatment in CRC. However, anti-PD-1 monotherapy alone has not yielded a clinical benefit in majority of these patients. Therefore, combination therapy is necessary to overcome this limitation. Previously, we have developed a FAPα-expressing tumor vaccine and demonstrated its anti-tumor effects by reversing of immunosuppressive tumor microenvironment (TME). In the current study, we hypothesized that combination therapy could enhance the effect of anti-PD1 therapy. Methods: FAPα-expressing CT26 cells were produced as previously described. To establish xenograft tumor model, CT26 cells were injected into left flank of BALB/c mice. Anti-PD-1 monoclonal antibodies(mAb) were injected intraperitoneally twice weekly up to 5 injections. FAPα-vaccines were injected subcutaneously around tumors weekly up to 3 injections. Histological sections of tumours were analysed by H&E staining and immunofluorescence. Flow cytometric analysis was done using an FACSCalibur (BD) and analyzed using FlowJo software (TreeStar). Gene expression profile in TME was analyzed by bulk RNA sequencing. Results: In mice xenograft CT26 tumor model, either anti-PD-1 immunotherapy or FAPα tumor cell vaccine produced suppression of CT26 tumor growth compared with the control group. However, combining anti-PD-1 mAb and FAPα-expressing tumor cell vaccine elicited the most remarkable anti-tumor effect and induced the most pronounced tumor growth suppression among all groups. In combination therapy group of the vaccine and PD-1 mAb, there were significantly more infiltrated lymphocytes and less FAPα+ fibroblasts. Flow cytometry demonstrated that the combination therapy promoted M1 macrophage polarization with a reduction of M2-like tumor associated macrophages (TAMs) and an increase of M1-like TAMs. This polarization was in consistent with the reduced level of IL-10 that is predominant cytokine secreted by M2 macrophages. A reduction of myeloid-derived suppressor cells (MDSCs) in the TME were also observed. Using bulk RNAseq we observed a set of immune related genes upregulated following combination therapy, including innate immune response, antigen presentation, lymphatic cells and recruitment and T cells activation. Decreased secretion of immune inhibitory chemokines (eg, CXCL12, IL-10, TGFβ) was also found by ELISA in tumors from mice treated with combination therapy. Conclusions: Our preliminary results suggested that combination therapy of FAPα-expressing tumor vaccine and anti-PD-1 therapy is a promising way to improve the efficacy of immunotherapy through modulation of immunosuppressive TME. In the future, more research is needed to further elucidate mechanism and clinical value of this combination approach.

Translational analysis of combination of 5-fluorouracil (FU), interferon (IFN)-alpha2, and nivolumab in unresectable fibrolamellar liver cancer.

e16198 Background: Fibrolamellar liver cancer (FLC) is a rare malignancy and involves multidisciplinary approaches including surgery, liver-directed, and systemic treatment. Combination of 5-fluorouracil (FU) and interferon (IFN)-a2 has long been used for FLC. We had a hypothesis that addition of anti-PD1 to 5FU+IFN-a2 may further improve clinical outcome and modulation of FLC tumor microenvironment. Methods: We treated 3 patients with 5FU+IFN-a2+nivolumab (N): 5FU 200 mg/m2 7-day-on/7-day-off and IFN-a2 (4 million U/m2 3 times/week while on 5-FU) for 8 weeks, followed by on-treatment biopsy and addition of N 480 mg every 4 weeks to 5FU+IFN-a2. Those who had conversion of unresectable disease to resectable one had the resected tumors analyzed. Gene expression profiling and immunohistochemistry (IHC) staining of immune cells were performed to investigate the evolution of tumor microenvironment on treatment. Results: 26 and 29 year-old patients had unresectable FLC with a 6.1 cm tumor in seg IV liver invading the common hepatic duct, and a 19.5 cm tumor, metastatic peritoneal nodules, and abdominal lymphadenopathy, respectively. After 9 and 5 months of therapy, unresectable tumors were converted to resectable ones with partial response (PR): the former had left hepatectomy and bile duct resection; the latter, left hepatectomy, resection of the omentum, spleen, left diaphragm, and lymph node dissection. A 20 year-old patient had multifocal liver tumors with peritoneal carcinomatosis and lymph nodes. He received treatment for 5 months and achieved stable disease, but hepatic tumor bleeding led to treatment discontinuation. 2 patients with PR had increased expression of genes related to T cells, CD8+T cells, Th1 cells, NK cells, cytotoxic cells, macrophages, dendritic cells, neutrophils, and B cells, subsequently on tumors before-treatment, on-treatment (5FU+IFN-a2), post-surgery (5FU+IFN-a2+N). IHC staining confirmed an increase in CD8+T cell infiltration after treatment with 5FU+IFN-a2 and 5FU+IFN-a2+N. The third patient whose disease was not converted to a resectable disease showed a decrease in genes related to Th1 cells, cytotoxic cells, dendritic cells, and neutrophils on-treatment biopsy (5FU+IFN-a2). This patient had a marginal increase in CD8+T cell infiltration on exposure to 5FU+IFN-a2. Conclusions: This clinical outcome of converting unresectable disease to resectable one suggests synergy of 5FU+IFN-a2 combined with N. The robust increased expression of genes related to immune system activation and increased infiltration of CD8+T cells on 5FU+IFN-a2+N may explain this clinical efficacy. Pro-inflammatory evolution of tumors on treatment could be used as predictive biomarkers. More prospective data are indicated to further confirm our hypothesis. Clinical trial information: NCT04380545 .

Microbubbles-assisted ultrasonication to promote tumor accumulation of therapeutics and modulation of tumor microenvironment for enhanced cancer treatments

Abnormal tumor vasculature is reported to severely hinder the therapeutic potency of diverse cancer therapeutics by restricting their intratumoral accumulation and/or causing therapeutic resistance. Herein, a microbubble-assisted ultrasonication technology (MAUT) of systemic administration of octafluoropropane-filled microbubbles together with tumor localized ultrasound (US) exposure is developed to generally promote intratumoral accumulation efficacy of three kinds of anti-tumor drugs with varying sizes through the cavitation effect-induced disruption of tumor blood vessels. MAUT was further shown to enable selective tumor hypoxia attenuation by filling microbubbles with high-purity oxygen and thus reducing the production of immunosuppressive lactic acids by suppressing glycolysis in cancer cells. Resultantly, MAUT markedly enhanced the therapeutic outcome of systemically administered anti-programmed death-1 (anti-PD-1) and chemotherapeutic doxorubicin (DOX) with and without using nanoscale liposomes as delivery vehicles. This work highlights that MAUT is a biocompatible yet versatile strategy to effectively reinforce the therapeutic potency of a broad range of cancer therapeutics, promising for future clinical usage.

Preclinical characterization of a novel anti-CD39/TGFβ-trap bispecific antibody that aims to modulate tumor microenvironment.

e14523 Background: Adenosine and TGF-β are two key immune suppressors in tumor microenvironment (TME) that cause broad immune suppression resulting in resistance to current CPI immunotherapies. Cancer cells, and other cell types within TME, frequently express TGFβ, which inhibits CD8+ T and Th1 cells and promotes epithelial–mesenchymal transition. We have designed a bi-specific targeting molecules blocking the two inhibitory pathways, aiming to create an immune-friendly TME. Methods: We have created a bifunctional antibody–ligand trap called ES014 that comprises an antibody targeting CD39 fused to a TGFβ receptor II ectodomain sequence. ES014 molecule could simultaneously disable CD39 to prevent extracellular ATP from degradation and neutralize autocrine/paracrine TGFβ in the vicinity of target cells. The immunological function of ES014 was studied in series of in vitro immuno-assays. The in vivo efficacy of ES014 was investigated in human PBMC engrafted models. The effects of ES014 on immune cells were also analyzed using malignant pleural effusions (MPE) collected from lung cancer patients. The pharmacokinetics (PK), pharmacodynamics (PD) and safety profile were assessed in cynomolgus monkeys after intravenous administration of ES014. Results: The bispecific antibody, ES014 exhibited synergistic effects in T cell activation and suppression of Treg differentiation. ES014 activates dendritic cells, macrophages and natural killer cells by maintaining extracellular ATP level and neutralizing the immune-suppressive effects of TGFβ. Interestingly, ES014 demonstrated a unique mechanism by protecting effector T cells from activation induced cell death (AICD), which was not observed with TGFβ receptor or anti-CD39 antibody alone, or the combination of the two. The ES014 molecule is effective in inhibiting tumor progression in a PD-1 antibody-unresponsive in vivo model. Treatment of ES014 in malignant pleural effusions (MPE) collected from lung cancer patients led to the switch of M2 to M1 macrophage as indicated by increased CD86 expression and decreased CD163 expression on CD11b+ cells. Importantly, more CD8+ T cells were maintained with ES014 treatment in MPE as compared to control, resulting in significant reduction of cancer number. ES014 was well tolerated in cynomolgus monkeys, and a phase I clinical study is ongoing to investigate the safety, tolerability, PK, PD and preliminary clinical activity of ES014 in patients with advanced solid tumors. Conclusions: ES014 is a first-in-class molecule that simultaneously targeting CD39 and TGFβ. ES014 counteracts TGFβ-mediated differentiation of Tregs and adenosine-induced immune tolerance, and protected effector T cells from AICD. Treatment of ES014 resulted in significant anti-tumor effects in ex vivo and in vivo efficacy models. A single agent activity of ES014 is therefore expected in clinical trials.

Effect of extended treatment with IAP inhibitor xevinapant post radiotherapy (RT) on efficacy and the tumor microenvironment (TME) in preclinical models.

6027 Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis, induced by RT and chemotherapy, and enhance antitumor immunity. In a randomized phase 2 study of patients with unresected locally advanced squamous cell carcinoma of the head and neck, xevinapant + chemoradiotherapy (CRT) significantly improved 18-month locoregional control and improved 3-year progression-free survival and 5-year overall survival vs placebo + CRT. A phase 3 confirmatory study (TrilynX NCT04459715) and a phase 3 study to evaluate xevinapant + RT in post-operative, high-risk, cisplatin-ineligible patients (XRay Vision NCT05386550) are ongoing. In these studies, we investigate 3 additional cycles of xevinapant or placebo monotherapy after completing combination treatment. Based on the role of IAPs in apoptosis, immunity, and stromal activation, continual dosing of xevinapant post RT may have additional therapeutic benefits. Methods: MC38 syngeneic tumor-bearing mice were treated with vehicle control, RT alone (3.6Gy once daily [QD], 5 days), or RT + xevinapant (100 mg/kg QD, 1, 2, or 4 weeks). To assess treatment-mediated TME modulation, immune cells in tumors were evaluated by fluorescence-activated cell sorting (FACS)–based immunophenotyping, and tumor-specific T cell responses were investigated by enzyme-linked immunosorbent spot (ELISpot) assays. To understand the effect of xevinapant on RT-induced cancer associated fibroblast (CAF) activation, CAF gene expression was profiled on cell lines and patient-derived primary cultures. Results: Xevinapant + RT increased antitumor efficacy compared with vehicle control or RT alone. Moreover, RT in combination with extended dosing of xevinapant for 4 weeks significantly improved therapeutic efficacy and prolonged survival compared with RT + shorter xevinapant dosing durations. FACS analysis demonstrated trends of increased numbers of CD8+ T cells, natural killer cells, and dendritic cells and decreased numbers of regulatory T cells in mice treated with xevinapant + RT. FACS and ELISpot further suggested an increase in antigen-specific T cell counts from baseline with combination therapy arm vs vehicle control or RT alone. Xevinapant may also suppress RT-mediated CAF activation, as indicated by downregulation of activation markers and CAF-derived secretory proteins. Conclusions: In preclinical models, extended dosing of xevinapant for 4 weeks post RT improved antitumor efficacy. Our data suggest that the effect may be, in part, modulated by TME responses, including enhanced antitumor immunity and suppressed pro-tumorigenic phenotype of CAFs. Based on these results, further evaluation is warranted to determine the mechanisms that underscore the therapeutic benefit offered by extended dosing of xevinapant.

Liquid Metal Nanoplatform Based Autologous Cancer Vaccines.

Therapeutic cancer vaccines have been vigorously sought to bolster host adaptive immunity against metastatic cancers, but tumor heterogeneity, ineffective antigen utilization, and immunosuppressive tumor microenvironment hinder their clinical applications. Autologous antigen adsorbability and stimulus-release carrier coupling with immunoadjuvant capacity are urgent for personalized cancer vaccines. Here, we propose a perspective strategy of using a multipotent gallium-based liquid metal (LM) nanoplatform for personalized in situ cancer vaccines (ISCVs). The antigen-capturing and immunostimulatory LM nanoplatform can not only effectively destroy orthotopic tumors to generate multifarious autologous antigens upon external energy stimulation (photothermal/photodynamic effect) but also capture and transport antigens into dendritic cells (DCs) to enhance antigen utilization (adequate DCs uptake, antigen-endo/lysosomal escape) and facilitate DCs activation (mimic alum immunoadjuvant capacity), which ultimately awaken systemic antitumor immunity (expand cytotoxic T lymphocytes and modulate tumor microenvironment). With immune checkpoint blockade (anti-PD-L1) to further relieve the immunosuppressive tumor microenvironment, the positive tumoricidal immunity feedback loop was established to effectively eliminate orthotopic tumors, inhibit abscopal tumor growth, relapse, and metastasis as well as tumor-specific prevention. Collectively, this study demonstrates the potential of a multipotent LM nanoplatform for personalized ISCVs, which will open frontier exploration of LM-based immunostimulatory biomaterials and may encourage further investigation of precise individualized immunotherapy.

Characterization of the metabolic alteration-modulated tumor microenvironment mediated by TP53 mutation and hypoxia

BackgroundTP53 mutation and hypoxia play an essential role in cancer progression. However, the metabolic reprogramming and tumor microenvironment (TME) heterogeneity mediated by them are still not fully understood. MethodsThe multi-omics data of 32 cancer types and immunotherapy cohorts were acquired to comprehensively characterize the metabolic reprogramming pattern and the TME across cancer types and explore immunotherapy candidates. An assessment model for metabolic reprogramming was established by integration of multiple machine learning methods, including lasso regression, neural network, elastic network, and survival support vector machine (SVM). Pharmacogenomics analysis and in vitro assay were conducted to identify potential therapeutic drugs. ResultsFirst, we identified metabolic subtype A (hypoxia-TP53 mutation subtype) and metabolic subtype B (non-hypoxia-TP53 wildtype subtype) in hepatocellular carcinoma (HCC) and showed that metabolic subtype A had an “immune inflamed” microenvironment. Next, we established an assessment model for metabolic reprogramming, which was more effective compared to the traditional prognostic indicators. Then, we identified a potential targeting drug, teniposide. Finally, we performed the pan-cancer analysis to illustrate the role of metabolic reprogramming in cancer and found that the metabolic alteration (MA) score was positively correlated with tumor mutational burden (TMB), neoantigen load, and homologous recombination deficiency (HRD) across cancer types. Meanwhile, we demonstrated that metabolic reprogramming mediated a potential immunotherapy-sensitive microenvironment in bladder cancer and validated it in an immunotherapy cohort. ConclusionMetabolic alteration mediated by hypoxia and TP53 mutation is associated with TME modulation and tumor progression across cancer types. In this study, we analyzed the role of metabolic alteration in cancer and propose a predictive model for cancer prognosis and immunotherapy responsiveness. We also explored a potential therapeutic drug, teniposide.

Antifibrotic Agent Mediated Tumor Microenvironment Modulation and Improved Nanomedicine Delivery in Solid Tumor.

The unique physiology of tumors limits the efficacy of chemotherapeutics. In efforts to improve the effectiveness of the existing chemotherapy drugs, nanomedicine emerged as a new hope but proved inadequate due to the transport barriers present within the tumor tissues, which limits the potential of nanomedicine. Dense collagen networks in fibrotic tissues contribute to hindering the penetration of molecular- or nano-scale medicine through tumor interstitium. In the present study, human serum albumin (HSA)-based nanoparticles (NPs) were developed for gemcitabine (GEM) and losartan (LST), which could offer secreted protein acids rich in cysteine (SPARC) and enhanced permeability and retention effect (EPR)-mediated drug accumulation in tumors. Also, the tumor microenvironment (TME) modulation approach using LST was coupled to investigate the impact on antitumor efficacy. GEM-HSA NPs and LST-HSA NPs were prepared by the desolvation-cross-linking method and characterized for size, potential, morphology, drug loading, drug-polymer interactions, and hemocompatibility. For investigating the efficacy of prepared NPs, cytotoxicity and mechanisms of cell death were elucidated in vitro by using various assays. Intracellular uptake studies of prepared HSA NPs indicated their uptake and cytoplasmic localization. Furthermore, in vivo studies demonstrated significantly improved anticancer efficacy of GEM-HSA NPs in combination with LST pretreatment. Extended LST treatment further improved the anticancer potential. It was shown that the improved efficacy of the nanomedicine was correlated with the reduced thrombospondin-1 (TSP-1) and collagen level in tumor tissue upon LST pretreatment. Moreover, this approach exhibited augmented nanomedicine accumulation in the tumor, and hematological, biochemical, and tissue histology indicated the safety profile of this combination regimen. Concisely, the undertaken study demonstrated the potential of the triple targeting (SPARC, EPR, TME modulation) approach for augmented efficacy of chemotherapeutics.

Antitumor Effects of a Distinct Sonodynamic Nanosystem through Enhanced Induction of Immunogenic Cell Death and Ferroptosis with Modulation of Tumor Microenvironment.

Sonodynamic therapy (SDT) holds great promise to be applied for cancer therapy in clinical settings. However, its poor therapeutic efficacy has limited its applications owing to the apoptosis-resistant mechanism of cancer cells. Moreover, the hypoxic and immunosuppressive tumor microenvironment (TME) also weakens the efficacy of immunotherapy in solid tumors. Therefore, reversing TME remains a formidable challenge. To circumvent these critical issues, we developed an ultrasound-augmented strategy to regulate the TME by utilizing an HMME-based liposomal nanosystem (HB liposomes), which can synergistically promote the induction of ferroptosis/apoptosis/immunogenic cell death (ICD) and initiate the reprograming of TME. The RNA sequencing analysis demonstrated that apoptosis, hypoxia factors, and redox-related pathways were modulated during the treatment with HB liposomes under ultrasound irradiation. The in vivo photoacoustic imaging experiment showed that HB liposomes enhanced oxygen production in the TME, alleviated TME hypoxia, and helped to overcome the hypoxia of the solid tumors, consequently improving the SDT efficiency. More importantly, HB liposomes extensively induced ICD, resulting in enhanced T-cell recruitment and infiltration, which normalizes the immunosuppressive TME and facilitates antitumor immune responses. Meanwhile, the HB liposomal SDT system combined with PD1 immune checkpoint inhibitor achieves superior synergistic cancer inhibition. Both in vitro and in vivo results indicate that the HB liposomes act as a sonodynamic immune adjuvant that is able to induce ferroptosis/apoptosis/ICD via generated lipid-reactive oxide species during the SDT and reprogram TME due to ICD induction. This sonodynamic nanosystem integrating oxygen supply, reactive oxygen species generation, and induction of ferroptosis/apoptosis/ICD is an excellent strategy for effective TME modulation and efficient tumor therapy.

Canine osteosarcoma as a solid tumor model for evaluation of dual valent B7H3-CXCR2 CAR T cell therapy

Abstract To develop more effective solid tumor CAR T cell therapies, we utilized the spontaneous canine metastatic osteosarcoma (OS) animal model. Our studies evaluated the relative activity of single B7H3 and dual-valent B7H3-CXCR2 CAR constructs. Studies were also designed to determine if modifying the tumor microenvironment (TME) with oral immunotherapy improved CAR activity, based on recent studies of losartan and propranolol in dogs with OS and glioma. We evaluated B7H3 expression on canine OS cell lines using flow cytometry and OS biopsies using IHC. High levels of B7-H3 were observed on OS cell lines and OS biopsies, whereas normal canine tissues were negative or low. To assess the utility of the dual CAR we determined that canine OS cell lines secreted high IL8 spontaneously. To assess the functionality of canine B7-H3 CAR T cells, we generated canine CAR T cells from whole blood from dogs with cancer. We next determined that canine dual CAR T cells exerted specific killing and cytokine activity against B7H3+ canine OS cell lines. To further establish functionality, we evaluated the two CAR constructs in a mouse xenograft model of canine OS. We found that the dual CAR construct was significantly more active than the single CAR construct in slowing tumor growth. We found that losartan and propranolol blocked migration of mouse inflammatory monocytes to xenografted canine OS tumors. Future steps will be to evaluate the effectiveness of dual CAR T cells with TME modification in dogs with metastatic OS, to investigate the translational relevance in human patients. Results from these studies in dogs will likely give more clinically relevant results for evaluating new CAR T cell therapies and bridging results between mouse models and human clinical trials. The V Foundation, The Shipley Family Foundation, and NIH T32 training grant

Hydrogen Sulfide Gas Amplified ROS Cascade: FeS@GOx Hybrid Nanozyme Designed for Boosting Tumor Chemodynamic Immunotherapy.

Chemodynamic immunotherapy that utilizes catalysts to produce reactive oxygen species (ROS) for killing tumor cells and arousing antitumor immunity, has received considerable attention. However, it is still restricted by low ROS production efficiency and insufficient immune activation, due to intricate redox homeostasis in tumor microenvironment (TME). Herein, a metalloprotein-like hybrid nanozyme (FeS@GOx) is designed by in situ growth of nanozyme (ferrous sulfide, FeS) in natural enzyme (glucose oxidase, GOx) to amplify ROS cascade for boosting chemodynamic immunotherapy. In FeS@GOx, GOx allows the conversion of endogenous glucose to gluconic acid and hydrogen peroxide, which provides favorable increasing hydrogen peroxide for subsequent Fenton reaction of FeS nanozymes, thus reinforcing the ROS production. Notably, H2 S release is activated by the gluconic acid generation-related pH decrease, which can suppress the activity of endogenous thioredoxin reductase and catalase to further inhibit the ROS elimination. Thus, FeS@GOx can sustainably amplify ROS accumulation and perturb intracellular redox homeostasis to improve chemodynamic therapy and trigger robust immunogenic cell death for effective immunotherapy combined with immune checkpoint blockade. This work proposes a feasible H2 S amplified ROS cascade strategy employing a bioinspired hybrid nanozyme, providing a novel pathway to multi-enzyme-mediated TME modulation for precise and efficient chemodynamic immunotherapy. This article is protected by copyright. All rights reserved.

Combination therapy with anti-programmed cell death 1 antibody plus angiokinase inhibitor exerts synergistic antitumor effect against malignant mesothelioma via tumor microenvironment modulation

Malignant pleural mesothelioma (MPM) is an asbestos-related fatal malignant neoplasm. Although there has been no reliable chemotherapeutic regimen other than combination therapy of cisplatin and pemetrexed for two decades, combination of ipilimumab plus nivolumab brought about a better outcome in patients with MPM. Thus, cancer immunotherapy using immune checkpoint inhibitor (ICI) is expected to play a central role in the treatment of MPM. To maximize the antitumor effect of ICI, we evaluated whether nintedanib, an antiangiogenic agent, could augment the antitumor effect of anti-programmed cell death 1 (PD-1) antibody (Ab). Although nintedanib could not inhibit the proliferation of mesothelioma cells in vitro, it significantly suppressed the growth of mesothelioma allografts in mice. Moreover, combination therapy with anti-PD-1 Ab plus nintedanib reduced tumor burden more dramatically compared with nintedanib monotherapy via inducing remarkable necrosis in MPM allografts. Nintedanib did not promote the infiltration of CD8+ T cells within the tumor when used alone or in combination with anti-PD-1 Ab but it independently decreased the infiltration of tumor-associated macrophages (TAMs). Moreover, immunohistochemical analysis and ex vivo study using bone marrow-derived macrophages (BMDMs) showed that nintedanib could polarize TAMs from M2 to M1 phenotype. These results indicated that nintedanib had a potential to suppress protumor activity of TAMs both numerically and functionally. On the other hand, ex vivo study revealed that nintedanib upregulated the expression of PD-1 and PD-ligand 1 (PD-L1) in BMDMs and mesothelioma cells, respectively, and exhibited the impairment of phagocytic activity of BMDMs against mesothelioma cells. Co-administration of anti-PD-1 Ab may reactivate phagocytic activity of BMDMs by disrupting nintedanib-induced immunosuppressive signal via binding between PD-1 on BMDMs and PD-L1 on mesothelioma cells. Collectively, combination therapy of anti-PD-1 Ab plus nintedanib enhances the antitumor activity compared with respective monotherapy and can become a novel therapeutic option for patients with MPM.

Extracellular Vesicle-Based Drug Delivery Systems for Head and Neck Squamous Cell Carcinoma: A Systematic Review.

It is estimated that there are over 890,000 new cases of head and neck squamous cell carcinoma (HNSCC) worldwide each year, accounting for approximately 5% of all cancer cases. Current treatment options for HNSCC often cause significant side effects and functional impairments, thus there is a challenge to discover more acceptable treatment technologies. Extracellular vesicles (EVs) can be utilized for HNSCC treatment in several ways, for example, for drug delivery, immune modulation, as biomarkers for diagnostics, gene therapy, or tumor microenvironment modulation. This systematic review summarizes new knowledge regarding these options. Articles published up to 11 December 2022, were identified by searching the electronic databases PubMed/MEDLINE, Scopus, Web of Science, and Cochrane. Only full-text original research papers written in English were considered eligible for analysis. The quality of studies was assessed using the Office of Health Assessment and Translation (OHAT) Risk of Bias Rating Tool for Human and Animal Studies, modified for the needs of this review. Of 436 identified records, 18 were eligible and included. It is important to note that the use of EVs as a treatment for HNSCC is still in the early stages of research, so we summarized information on challenges such as EV isolation, purification, and standardization of EV-based therapies in HNSCC.

The Diet as a Modulator of Tumor Microenvironment in Colorectal Cancer Patients.

Colorectal cancer (CRC) is one of the most common cancers in Western countries and remains the second most common cause of cancer death worldwide. Many studies show the importance of diet and lifestyle in the incidence of CRC, as well as in CRC prevention. However, this review summarizes those studies that analyze the impact of nutrition on tumor microenvironment modulation and cancer progression. We review the available information about the effects of specific nutrients on cancer cell progression and on the different cells within the tumor microenvironment. Diet and nutritional status in the clinical management of colorectal cancer patients are also analyzed. Finally, future perspectives and challenges are discussed, with a view to improving CRC treatments by employing nutritional approaches. These promise great benefits and will eventually improve CRC patients' survival.

Abstract LB303: Tumor microenvironment modulation by SERPINE1 increases radioimmunotherapy in murine model of gastric cancer

Abstract The deposition of the extracellular matrix (ECM) is associated with poor prognosis and therapy response in gastric cancer. An elevated extracellular matrix (ECM) and interstitial fluid pressure in gastric cancer limits the targeting of HER2-expressing gastric cancer cells when radioimmunotherapy (RIT) with 64Cu-trastuzumab (64Cu-TRZ) is utilized. Here we used Losartan (LOS), which is an antihypertensive drug, to downregulate ECM and interstitial fluid pressure in gastric cancer mice model. After 40 mg/kg of LOS treatment, a 2-fold higher Alexa-647-TRZ uptake significantly enhanced 64Cu-TRZ uptake by positron emission tomography (PET) imaging, LOS treatment decreased the tumor volume, and enhanced the survival rate. LOS-treated samples showed reduced SERPINE1 mRNA expression compared with the controls, as determined by qRT-PCR. Downregulated mRNA expression of TGFβ-1, and upregulated mRNA expression of MMP-2 in LOS-treated samples were observed. The corresponding protein expression of SERPINE1 was confirmed using western blotting. We also confirmed that silencing of SERPINE1 increased MMP2 activity. We revealed that the enhanced therapeutic effect of 64Cu-TRZ via LOS was due to the downregulation of SERPINE1, which degrades the ECM through MMP2. Our novel combinational therapy of using 64Cu-TRZ with LOS is highly effective for treatment of gastric cancer. Citation Format: Javeria Zaheer, Joycie Shanmugiah, Hyeongi Kim, Jin Su Kim. Tumor microenvironment modulation by SERPINE1 increases radioimmunotherapy in murine model of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB303.

Abstract 657: IRAK1 regulated inflammatory signal from the leukemia cells modulates tumor microenvironment and promote leukemogenesis

Abstract Hematologic malignancy associated with fibroblast growth factor receptor 1 (FGFR1) translocations develops an aggressive stem cell leukemia/lymphoma (SCLL), 80% of which progress to AML and only ~15% show survival of > 15 months. In addition, 23.6% of de novo AML patients overexpress FGFR1, compared with healthy individuals. Therefore, investigating how tumor-intrinsic FGFR1 signaling modulates the tumor microenvironment and their contribution to leukemogenesis is not only important for understanding leukemia initiation and progression but also critical for the development of an efficient cure for AML patients. Interleukin Receptor Associated Kinase 1 (IRAK1) is a protein kinase that initiates a cascade of signaling events eventually leading to activation of proinflammatory genes, including IL-6, IL-10, TNF-α, and IFN-γ, which play critical roles in inflammation and innate immunity. The role of IRAK1 has been reported in several different subtypes of leukemia, including B cell-like diffuse large B-cell lymphoma (ABC DLBCL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and melanoma. Most previous IRAK1 studies, however, only focused on its survival and proliferation promoting roles inside the tumor cell itself. Since most of these studies engrafted human cancer cell lines into NSG mice, which lack a functional immune system, it was impossible to investigate the function of IRAK1 in modulating the tumor microenvironment. Using our well-established syngeneic SCLL mouse model, we defined IRAK1 gene as a critical target of FGFR1 in driving leukemogenesis. Our preliminary studies revealed that IRAK1 knockout (KO) in SCLL leukemia cells leads to T cell mediated clearance of the leukemia cells. In contrast, IRAK1 expressing mock control (MC) cells induce the accumulation of myeloid derived suppressor cells (MDSCs), which sequentially suppress T cell function to promote leukemia progression. These observations indicate a critical function for IRAK1 in modulating the tumor microenvironment and promoting leukemogenesis. Most importantly, pilot cytokine profiling of the MC and IRAK1 KO leukemia cells identified IFN-γ as one of the direct targets of IRAK1 in leukemia cells, and scRNA-Seq analysis revealed predominant expression of IFNGR1 and identified activation of the IFN-γ signaling pathway in leukemia induced MDSCs. Genes in this pathway include IFN-γ directly regulated genes, such as CD274 and SOCS3. Further engraftment using either IFNGR1-/- mice or IFNG KO leukemia cells revealed the involvement of this leukemia cell intrinsic, IRAK1 regulated IFN-γ in modulating the tumor microenvironment to promote leukemogenesis. In summary, our data suggests that leukemia cell-derived, IRAK1 regulated inflammatory signals act as critical modulators in establishing a tumor favorable microenvironment during leukemogenesis. Citation Format: Ting Zhang, Xiaocui Lu, Atsuko Matsunaga, Xuexiu Fang, Stephanie Fay Mori, John Cowell, Tianxiang Hu. IRAK1 regulated inflammatory signal from the leukemia cells modulates tumor microenvironment and promote leukemogenesis [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 657.