Changes In Tumor Microenvironment Research Articles

Diffuse Reflectance Spectroscopy of Changes in Tumor Microenvironment in Response to Different Doses of Radiation.

Radiation therapy plays an important role in cancer treatment, as it is an established method used as part of the treatment plan for the majority of cancer patients. Real-time monitoring of the effects of radiation on the tumor microenvironment can contribute to the development of better treatment plans. In this study, we use diffuse reflectance spectroscopy, a non-invasive optical fiber-based technique, to determine the effects of different doses of radiation on the tumor microenvironment, as well as to determine the sensitivity of diffuse reflectance spectroscopy to low doses of radiation that are used in the treatment of certain cancers. We injected 4T1 cells into 50 Balb/c mice to generate tumor xenografts. When the tumors grew to 200 mm3, we distributed the mice into a control group or one of three radiation groups: 1, 2, or 4 Gy/fraction, and they underwent treatment for five consecutive days. We measured the tumor volume and collected diffuse reflectance spectra every day, with optical measurements being acquired both before and one h postirradiation on the five days of treatment. Based on the diffusely reflected light, we quantified vascular oxygenation, total hemoglobin content, and tissue scattering within these tumors. There was a significant increase in tumor vascular oxygenation, which was primarily due to an increase in oxygenated hemoglobin, in response to a 1 Gy/fraction of radiation, while there was a decrease in tissue scattering in response to all doses of radiation. Immunohistochemical analysis revealed that tumor cell proliferation and apoptosis were higher in irradiated groups compared to the control group. Our findings show that diffuse reflectance spectroscopy is sensitive to microenvironmental changes in tumors treated with doses of radiation as low as 1 Gy/fraction.

S473 Changes in Tumor Micro-Environment With Liquid Nitrogen Spray Cryotherapy and Chemoradiation in Locally Advanced Esophageal Cancer

S473 Changes in Tumor Micro-Environment With Liquid Nitrogen Spray Cryotherapy and Chemoradiation in Locally Advanced Esophageal Cancer

Combination approaches in hepatocellular carcinoma: How systemic treatment can benefit candidates to locoregional modalities.

The management of hepatocellular carcinoma (HCC) is challenging because most patients have underlying cirrhosis, and the treatment provides, historically, a limited impact on the natural history of patients with advanced-stage disease. Additionally, recurrence rates are high for those patients who receive local and locoregional modalities, such as surgical (resection and transplantation) or image-guided (ablation and intra-arterial) therapies. Translational research has led to new concepts that are reshaping the current clinical practice. Substantial advancements were achieved in the understanding of the hallmarks that drive hepatocarcinogenesis. This has primed a successful incorporation of novel agents with different targets, such as anti-angiogenic drugs, targeted-therapies, and immune-checkpoint inhibitors. Although clinical trials have proven efficacy of systemic agents in advanced stage disease, there is no conclusive evidence to support their use in combination with loco-regional therapy. While novel local modalities are being incorporated (e.g., radioembolization, microwave ablation, and irreversible electroporation), emerging data indicate that locoregional treatments may induce tumor microenvironment changes, such as hyperexpression of growth factors, release of tumor antigens, infiltration of cytotoxic lymphocytes, and modulation of adaptative and innate immune response. Past trials that evaluated the use of antiangiogenic drugs in the adjuvant setting after ablation or chemoembolization fail to demonstrate a substantial improvement. Current efforts are directed to investigate the role of immunotherapy-based regimens in this context. The present review aims to describe the current landscape of systemic and locoregional treatments for HCC, present evidence to support combination approaches, and address future perspectives.

Analysis of the Effect of SNAI Family in Breast Cancer and Immune Cell.

SNAI family members are transcriptional repressors that induce epithelial-mesenchymal transition during biological development. SNAIs both have tumor-promoting and tumor-inhibiting effect. There are key regulatory effects on tumor onset and development, and patient prognosis in infiltrations of immune cell and tumor microenvironmental changes. However, the relationships between SNAIs and immune cell infiltration remain unclear. We comprehensively analyzed the roles of SNAIs in cancer. We used Oncomine and TCGA data to analyze pan-cancer SNAI transcript levels. By analyzing UALCAN data, we found correlations between SNAI transcript levels and breast cancer patient characteristics. Kaplan–Meier plotter analysis revealed that SNAI1 and SNAI2 have a bad prognosis, whereas SNAI3 is the opposite. Analysis using the cBio Cancer Genomics Portal revealed alterations in SNAIs in breast cancer subtypes. Gene Ontology analysis and gene set enrichment analysis were used to analyze differentially expressed genes related to SNAI proteins in breast cancer. We used TIMER to analyze the effects of SNAI transcript levels, mutations, methylation levels, and gene copy number in the infiltration of immune cell. Further, we found the relationships between immune cell infiltration, SNAI expression levels, and patient outcomes. To explore how SNAI proteins affect immune cell, we further studied the correlations between immunomodulator expression, chemokine expression, and SNAI expression. The results showed that SNAI protein levels were correlated with the expression of several immunomodulators and chemokines. Through analysis of PharmacoDB data, we identified antitumor drugs related to SNAI family members and analyzed their IC50 effects on various breast cancer cell lines. In summary, our study revealed that SNAI family members regulate different immune cells infiltrations by gene copy number, mutation, methylation, and expression level. SNAI3 and SNIA1/2 have opposite regulatory effects. They all play a key role in tumor development and immune cell infiltration, and can provide a potential target for drug therapy.

Differential OAT methylation correlates with cell infiltration in tumor microenvironment and overall survival postradiotherapy in oral squamous cell carcinoma patient.

Given that DNA methylation and tumor microenvironment (TME) are susceptible to radiotherapy, we aimed to figure out specific differential DNA methylation to reflect oral squamous cell carcinoma (OSCC) prognosis and associated effect on TME changes postradiotherapy, performing as an efficient biomarker. Differentially methylation analysis was performed using data from The Cancer Genome Atlas. Curves of Kaplan-Meier (K-M) survival, cumulative hazard and events, Cox proportional hazards, and linear regression model were conducted to screen and validate differential methylation genes, while multiple regression equation to analyze if ornithine aminotransferase (OAT) methylation correlates with radiotherapy. For correlation between OAT methylation and immune infiltrates, CIBERSORT and ESTIMATE algorithms were performed, following gene set enrichment analysis (GSEA) and ssGSEA analysis to evaluate biological process. Compared to normal tissues, only OAT in OSCC was differential significantly by K-M analysis (p=0.0364). OAT hypermethylation was associated with increased overall survival (hazard ratio: 0.65, p=0.0358). Radiotherapy correlated with OAT methylation (β=-0.01, p=0.0061); most patients with OAT hypermethylation were radiation-sensitive. Hypomethylated OAT correlated with higher cell infiltrations in TME. Neuroactive ligand-receptor interaction was most significantly related to OAT methylation (p=9.2e-10). Sulfur metabolism was the most significantly in OAT hypermethylation group (p=0.0041) and RIG-I-like receptor in OAT hypomethylation group (p=0.0094). OAT methylation can serve as a predictor of OSCC prognosis postradiotherapy with potential mechanism by changing cell infiltrations in TME, but further experimental study deserves to carry out confirming the role and mechanism of OAT methylation in OSCC.

Dual-Mode Tumor Imaging Using Probes That Are Responsive to Hypoxia-Induced Pathological Conditions.

Hypoxia in solid tumors is associated with poor prognosis, increased aggressiveness, and strong resistance to therapeutics, making accurate monitoring of hypoxia important. Several imaging modalities have been used to study hypoxia, but each modality has inherent limitations. The use of a second modality can compensate for the limitations and validate the results of any single imaging modality. In this review, we describe dual-mode imaging systems for the detection of hypoxia that have been reported since the start of the 21st century. First, we provide a brief overview of the hallmarks of hypoxia used for imaging and the imaging modalities used to detect hypoxia, including optical imaging, ultrasound imaging, photoacoustic imaging, single-photon emission tomography, X-ray computed tomography, positron emission tomography, Cerenkov radiation energy transfer imaging, magnetic resonance imaging, electron paramagnetic resonance imaging, magnetic particle imaging, and surface-enhanced Raman spectroscopy, and mass spectrometric imaging. These overviews are followed by examples of hypoxia-relevant imaging using a mixture of probes for complementary single-mode imaging techniques. Then, we describe dual-mode molecular switches that are responsive in multiple imaging modalities to at least one hypoxia-induced pathological change. Finally, we offer future perspectives toward dual-mode imaging of hypoxia and hypoxia-induced pathophysiological changes in tumor microenvironments.

Dissecting the Single-Cell Transcriptome Network of Immune Environment Underlying Cervical Premalignant Lesion, Cervical Cancer and Metastatic Lymph Nodes.

Cervical cancer (CC) is one of the most common malignancy in women worldwide. It is characterized by a natural continuous phenomenon, that is, it is in the initial stage of HPV infection, progresses to intraepithelial neoplasia, and then develops into invasion and metastasis. Determining the complexity of tumor microenvironment (TME) can deepen our understanding of lesion progression and provide novel therapeutic strategies for CC. We performed the single-cell RNA sequencing on the normal cervix, intraepithelial neoplasia, primary tumor and metastatic lymph node tissues to describe the composition, lineage, and functional status of immune cells and mesenchymal cells at different stages of CC progression. A total of 59913 single cells were obtained and divided into 9 cellular clusters, including immune cells (T/NK cells, macrophages, B cells, plasma cells, mast cells and neutrophils) and mesenchymal cells (endothelial cells, smooth muscle cells and fibroblasts). Our results showed that there were distinct cell subpopulations in different stages of CC. High-stage intraepithelial neoplasia (HSIL) tissue exhibited a low, recently activated TME, and it was characterized by high infiltration of tissue-resident CD8 T cell, effector NK cells, Treg, DC1, pDC, and M1-like macrophages. Tumor tissue displayed high enrichment of exhausted CD8 T cells, resident NK cells and M2-like macrophages, suggesting immunosuppressive TME. Metastatic lymph node consisted of naive T cell, central memory T cell, circling NK cells, cytotoxic CD8+ T cells and effector memory CD8 T cells, suggesting an early activated phase of immune response. This study is the first to delineate the transcriptome profile of immune cells during CC progression using single-cell RNA sequencing. Our results indicated that HSIL exhibited a low, recently activated TME, tumor displayed immunosuppressive statue, and metastatic lymph node showed early activated phase of immune response. Our study enhanced the understanding of dynamic change of TME during CC progression and has implications for the development of novel treatments to inhibit the initiation and progression of CC.

A Hypoxia-Sensitive Drug Delivery System Constructed by Nitroimidazole and its Application in the Treatment of Hepatocellular Carcinoma.

Hypoxia is an important pathological phenomenon, and it can induce many tumor microenvironment changes, such as accumulations of intracellular lactic acid, decrease of tumor microenvironment pH value, and regulate a series of physiological and pathological processes such as adhesion, metastasis, and immune escape. Hypoxic tumor cells act as a key target for treating tumor. In this research, we designed and prepared PEG-nitroimidazole grafts, PEG-NI, and FA-PEG-NI. We first explored their physical and chemical properties to serve as a drug carrier. Then, the hypoxia-sensitive properties such as particle size changes and drug release were investigated. Finally, the tumor targeting ability was studied in vitro and in vivo, and anti-tumor capacity was determined. Both grafts showed excellent property as a nanodrug carrier and showed favorable drug encapsulation ability of sorafenib with the help of the hydrophobic chain of 6-(BOC-amino) hexyl bromide. The micelles responded to the hypoxic tumor environment with chemical and spatial structure changes leading to sensitive and fast drug release. With the modification of folic acid, FA-PEG-NI gained tumor targeting ability in vivo. FA-PEG-NI graft proved a potential targeting drug delivery system in the treatment of hypoxic hepatocellular carcinoma.

Abstract 1943: Elucidating early tumor microenvironmental changes due to immunotherapy with label-free Raman spectroscopy and machine learning

Abstract Clinical benefit is observed in only a limited fraction of patients that undergo immunotherapy for a variety of cancer types. Therefore, the determination of robust biomarkers for accurate prediction of response to immunotherapeutic agents remains one of the grand challenges of 21st century. Our primary goal was to investigate if phenotypic differences due to treatment with ICI can be recognized with minimally perturbative molecular tools and guide personalization of immunotherapy. In this work, we provide first-in-class evidence that Raman spectroscopy (an optical method) and machine learning allows sensitive detection of early changes in biomolecular composition of tumors in response to immunotherapy with immune checkpoint inhibitors (ICI). By studying the widely investigated CT26 murine model of colorectal cancer treated with anti-CTLA-4 (n = 8) and anti-PD-L1 (n = 7) ICIs and controls (n = 10), we revealed new biological insights into the nature and degree of microenvironmental modifications induced by exposure to clinically relevant doses of ICI. Multivariate curve resolution-alternating least squares (MCR-ALS) decomposition of Raman spectral datasets revealed early changes in lipid, nucleic acid, and collagen content of the tumors following therapy. We trained supervised classification models - support vector machines and random forests on the spectral datasets and showed that the models provided excellent prediction accuracies for response to both ICIs and delineated spectral markers specific to each therapy, consistent with their differential mechanisms of action. On the basis of these findings, we sought to determine if the tumor microenvironment changes delineated by Raman spectroscopy correspond to proteomic alterations via quantitative mass spectrometry. Of the more than 6600 proteins identified, about 136 proteins were found to be significantly different in treated tumors relative to controls (p < 0.05 and log2 fold change of > 2). A subset of these differentially expressed proteins is known to regulate lipid metabolism and extracellular matrix composition, while others are known to control transcriptome dynamics or are associated with response to ICI therapy, thereby corroborating our Raman spectroscopic measurements. Our observation of biomolecular changes in the TME should catalyze detailed investigations for translating such markers and label-free Raman spectroscopy for clinical monitoring of immunotherapy response in cancer patients. Citation Format: Santosh K. Paidi, Joel R. Troncoso, Narasimhan Rajaram, Ishan Barman. Elucidating early tumor microenvironmental changes due to immunotherapy with label-free Raman spectroscopy and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1943.

Abstract 6132: Synchrotron Fourier-transform infrared microspectroscopy: Characterization of tumor infiltrating monocytes stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells

Abstract Background: Many studies suggested that the pathogenesis of inflammatory breast cancer (IBC) is related to inflammatory manifestations and changes in tumor microenvironment (TME). IBC characterized by significantly higher infiltration of tumor associated macrophages (TAMs). TAMs contribute to the metastatic process in IBC patients via secreting many cytokines such as TNF, IL-6, IL-8, and IL-10 that enhance invasion and angiogenesis. To understand the role of TAMs in IBC, there is a need to firstly understand how IBC TME affect the polarization of tumor infiltrated monocytes via using sensitive and accurate analytical tool. Synchrotron FTIR microspectroscopy (SR-µFTIR) is a highly advanced analytical approach has the ability to detect any biochemical changes even prior to any morphological changes. Herein we will use SR-µFTIR to study the biochemical changes in tumor infiltrating monocytes after stimulation by the secretome of non-IBC and IBC breast cancer cells. Methods: ten breast cancer patients were enrolled in this study (5 IBC and 5 non-IBC). tumor infiltrated monocytes were isolated from patient’s blood samples which collected during modified radical mastectomy surgery. The isolated monocytes were stimulated with the secretome of MCF7, MDA-MB-231 and SUM149 cell line. Synchrotron FTIR Microspectroscopy (SR-FTIRM) single cell analysis was done at SESAME light source. The obtained raw spectral data were processed and analyzed using OMNIC©, PeakFit© V4.12, and Unscrambler© X v.10.4 software. Results: The obtained FTIR spectra showed very intense absorption bands in the region of 1700- 1500 cm-1 attributed to the amide I (ν(C=O), & νAS (C-N), δ (N-H) and amide II ν(C-N), δ (N-H) proteins bands, with a strong broad signal in the 3600-3200 cm-1 related to the νN-H Amides A and B. In addition, three peaks of different intensity and area were detected in the lipids region of the vCH2 and vCH3 stretching modes positioned within the 3000 - 2800 cm-1 range. Curve fitting assignment clearly demonstrate some differences between the three stimulated monocytes in both amide I and II, and lipids regions. The Principal Component Analysis (PCA) scores plot of the second derivative spectra of the amide region explain 77% of the variance, with 63% explained by PC1 and 14% explained by PC2. On the other hand, the PCA scores plot of the lipid region explain a total of 92% of the variance with PC1 explaining 72% and PC2 explaining 20%. Conclusion: The obtained results showed that there is different effect of non-IBC and IBC cells on the polarization of tumor infiltrating monocytes even with the cell surface markers look the same and from this, we may start to understand different roles of TAMs in IBC microenvironment. Citation Format: Hossam T. Mohamed, Gihan Kamel, Aya A. El-Sharkawy, Noura El-Husseiny, Mohamed El-Shinawi, Mona M. Mohamed. Synchrotron Fourier-transform infrared microspectroscopy: Characterization of tumor infiltrating monocytes stimulated by the secretome of inflammatory and non-inflammatory breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6132.

Abstract CT010: A phase II study of durvalumab and tremelimumab with front-line neoadjuvant chemotherapy in patients with advanced-stage ovarian cancer (KGOG 3046/TRU-D)

Abstract KGOG 3046 is a single-arm phase 2 study evaluating the combination of dual immune checkpoint inhibition (durvalumab [D] and tremelimumab [T]) with neoadjuvant chemotherapy (NAC) for the upfront advanced-stage epithelial ovarian cancer (aEOC). This study enrolled patients with FIGO stage IIIC-IV EOC. Patients were assigned to one of the following neoadjuvant chemo-immunotherapy (NACI): original cohort arm1 (D 1,500 mg q3w + T 75 mg q3w + paclitaxel 175 mg/m2 + carboplatin AUC 5 [3 cycles]) or expansion cohort arm2 (D 1,500 mg q3w + T 300 mg [1 dose] + same chemotherapy as original). Arm2 was initiated after the completion of arm1. After NAC, all patients underwent interval debulking surgery (IDS). After IDS, three cycles of D (1,120 mg) and adjuvant chemotherapy followed by D maintenance (1,120 mg [total 12 cycles]) were administered. During treatment, serial biopsies were performed to explore immunologic changes in tumor microenvironment (TME). The primary endpoint was a 12-month PFS rate. Interim analysis was performed to evaluate outcomes after NAC after all patients underwent IDS. A total of 45 patients were enrolled as follows: arm1 (n=23) and arm2 (n=22). The majority of the patients presented with high-grade serous carcinoma (91.1%) and stage IV disease (77.8%). After NAC, the ORR was 86.7% by RECIST v1.1 (95.7% in arm1 and 81.8% in arm2, P=0.17). At IDS, 30 patients (66.7%) had R0 resection (73.9% in arm1 and 59.1% in arm2, P=0.353). Fourteen patients (31.1%) had a CRS 3 (39.1% in arm1 and 22.7% in arm2, P=0.337) and 5 (11.1%) had a pathologic remission. Skin rashes were the most common adverse events (51.1%) and grade ≥3 events occurred in 3 patients (15.6%), which completely resolved after steroid use. Stromal TIL (P=0.0335), CD8 (P<0.001), CD8/Foxp3 ratio (P<0.001) on immunohistochemistry were significantly increased after NACI. When TILs were analyzed in detail by flow cytometry, suppressive intratumoral regulatory T cells (Treg) were significantly decreased after NACI in both arm (P<0.05). Interestingly, the frequency of intratumoral Treg after NACI was lower in arm1 than arm2 (P<0.05). These interim data highlight the clinical activity and manageable toxicity profile for the addition of D and T to NAC in aEOC. Additional translational study data will be presented at the meeting. Summary of secondary outcomes D+T75+CT (n=23) D+T300+CT (n=22) P value ORR by RECIST 22 (95.7%) 18 (81.8%) 0.187 ORR by PERCIST 13/14a (92.9%) 16/18a (88.9%) 0.856 R0 at IDS 17 (73.9%) 13 (59.1%) 0.353 CRS3 at IDS 9 (39.1%) 5 (22.7%) 0.337 Pathologic CR 4 (17.4%) 1 (4.5%) 0.348 a, PERCIST was available in 14 (original) and 18 (expansion) cohorts. Citation Format: Junsik Park, Eunhyang Park, Je-Gun Joung, Myong Cheol Lim, Byoung-Gie Kim, Jae Weon Kim, Jung Bok Lee, Sunghoon Kim, Chel Hun Choi, Hee Seung Kim, Sang Yoon Park, Jongsuk Chung, Jung-Yun Lee. A phase II study of durvalumab and tremelimumab with front-line neoadjuvant chemotherapy in patients with advanced-stage ovarian cancer (KGOG 3046/TRU-D) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT010.

Abstract CT502: Preliminary immunological monitoring of first-in-human immunotherapy-trio for advanced head and neck squamous cell carcinoma

Abstract Background: Preclinical studies document that complex cancer vaccines, combined with T cell agonists and anti-PD-1, can augment therapeutic efficacy. Here we report preliminary immunological analyses of patients enrolled in a first-in-human immunotherapy-trio study of multivalent autophagosome vaccine (DPV-001), with sequenced checkpoint inhibition (anti-PD-1; retifanlimab), with/without anti-GITR agonist (INCAGN-1949), in recurrent or metastatic HNSCC (NCT04470024). Methods: Peripheral blood (PB) and sera are collected regularly and PB are evaluated by flow cytometry. Biopsies obtained at baseline, D15 and D45 are analyzed by multiplex IF and 10x Genomics scRNA-Seq. Sera are being analyzed by phage immunoprecipitation (PhIP) sequencing for reactivity against the human proteome. Results: Preliminary results document increases in activated CD4 and CD8 effector memory T cells (TEM) with vaccine alone. Changes in tumor microenvironment (TEM) were also observed with increased infiltration of immune cells. Evaluation of changes to humoral immunity, T cell function and TCR analyses are ongoing. Conclusions: We previously reported immunological monitoring of a phase I/II trial of an autophagosome cancer vaccine (DPV-001) containing more than 300 shared cancer antigens, as adjuvant therapy for NSCLC. Vaccination induced or augmented immune responses to more than 50 cancer antigens shared with head and neck squamous cell carcinoma (HNSCC). Preclinical studies combining this cancer vaccine with αGITR agonist and αPD-1 augmented therapeutic efficacy [PMID: 31747946], and provided the rationale for the current study. This is the first clinical trial to perform such a study with αGITR agonist (INCAGN-1949), in humans. Support: Incyte, Providence Medical Foundation, The Harder Family, Nancy Lematta. Citation Format: Rom S. Leidner, Matthew H. Taylor, Tarsem L. Moudgil, Tanisha L. Christie, Yoshinobu Koguchi, Alexa Dowdell, William L. Redmond, Shawn M. Jensen, Carmen Ballesteros-Merino, Jake A. Vancampen, Venkatesh Rajamanickam, Brady M. Bernard, Christopher Paustian, Traci L. Hilton, Hong-Ming Hu, Richard B. Bell, Walter J. Urba, Carlo B. Bifulco, Brian Piening, Bernard A. Fox. Preliminary immunological monitoring of first-in-human immunotherapy-trio for advanced head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT502.

Abstract 5974: Multimodal molecular imaging detects early reoxygenation induced by hyaluronan depletion in pancreatic cancer model mouse

Abstract [Purpose] In pancreatic ductal adenocarcinoma (PDAC) which is characterized by an intense desmoplastic feature, the extracellular matrix (ECM) can significantly influence the tumor microenvironment (TME). Hyaluronan (HA), a major component of ECM, is associated with elevated tumor pressure, vascular collapse, and poor perfusion in TME, conferring hypoxia. HA expression is also correlated with poor prognosis in the patients with PDAC. PEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan in tumors. The resultant improvement in vascular patency and blood perfusion is expected to increase the delivery of therapeutic molecules. The aim of this study was to investigate the change in physiologic and metabolic profile of the tumor in response to treatment with PEGPH20 using multi-modal imaging techniques. We also investigated the capability of PEGPH20 to enhance treatment effect of radiation. [Methods] Athymic nude mice were inoculated with BxPC3 (human pancreatic adenocarcinoma) tumor cells transduced with hyaluronan synthase 3 (HAS3) to the right tibial periosteum. BxPC3-HAS3 tumor treated with PEGPH20 or control buffer were scanned with Electron paramagnetic Resonance imaging (EPRI), dynamic contrast enhanced (DCE) MRI, ultra-small superparamagnetic iron oxide (USPIO) MRI, Photoacoustic imaging (PAI), and Hyperpolarized 13C-MRI using [1-13C] pyruvate to evaluate intratumor pO2, intratumor perfusion, blood volume, O2 saturation, and glycolysis, respectively. [Results] EPRI showed significantly increased pO2 in PEGPH20 treated group. DCE-MRI and USPIO-MRI showed improved perfusion/permeability and local blood volume, respectively after PEGPH20 treatment, accounting for the increase in tumor oxygenation. PAI provided the evidence of immediate changes in tumor oxygenation after treatment. Hyperpolarized 13C-MRI using [1-13C] pyruvate suggested the decreased glycolytic flux evaluated by lactate/pyruvate ratio after PEGPH20 treatment. Combination of radiotherapy and PEGPH20 synergistically delayed tumor progression and prolonged the survival. [Conclusions] This study examined the effect of PEGPH20 on TME in PDAC xenograft model by using non-invasive multimodal imaging techniques. In summary, the non-invasive imaging modalities were useful in evaluating the changes in hemodynamics and metabolism in TME induced by modulation of ECM such as PEGPH20 treatment. PEGPH20 enhanced treatment effect of radiation therapy. The results validated the utility of the imaging methods to non-invasively monitor the changes in TME and predicted the radiosensitizing effect of hyaluronan depletion. Citation Format: Yu Saida, Tomohiro Seki, Shun Kishimoto, Jeffrey R. Brender, Gadisetti VR. Chandramouli, Yasunori Otowa, Kota Yamashita, Kazutoshi Yamamoto, Nallathamby Devasahayam, Murali C. Krishna. Multimodal molecular imaging detects early reoxygenation induced by hyaluronan depletion in pancreatic cancer model mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5974.

Abstract CT145: Systemic rintatolimod and interferon-α2b selectively reprogram local tumor microenvironment in patients with metastatic triple negative breast cancer for enhanced influx of cytotoxic T-lymphocytes but not regulatory T-cells

Abstract Background: Effective immune therapies depend on the presence of cytotoxic T-lymphocytes (CTLs) in the tumor microenvironment (TME). Our preclinical data showed synergy between TLR3 ligands and interferon-α (IFN-α) in reprogramming the TME, but not healthy tissues, to selectively enhance CTL attraction, providing rationale for their systemic application to enhance local CTL densities in “cold” tumors. The pilot study NCT03599453 evaluated the safety of systemic chemokine modulating regimen (CKM) composed of i.v. rintatolimod (Ampligen; selective TLR3 ligand) and IFN-α, and its ability to promote local CTL influx to mTNBC lesions. Methods: Six evaluable patients (33-75 years) with mTNBC received 6 doses of rintatolimod (200 mg i.v.), IFN-α (INTRON-A; 20MU/m2 i.v.) and COX-2 inhibitor (celecoxib; 2 x 200 mg, p.o.) over 2 weeks, with tumor biopsies obtained before (within 6 days) and after (within 5 days) CKM. All patients received follow-up pembrolizumab (200 mg, i.v, Q3 weeks). The primary endpoint was the change in the CTL marker CD8α in the TME with a planned interim analysis after 3 patients (α=0.03) and final analysis after 6 patients (α=0.084). Correlative studies analyzed additional markers of CTLs, regulatory T-cells (Tregs), and CTL- and Treg-attracting chemokines in the TME and blood. Results: Treatment was well tolerated with mostly grade 1/2 adverse events and one grade 3 clinically significant pneumonitis and immune thrombocytopenic purpura observed during follow up pembrolizumab treatment. We observed uniform increases of intratumoral type-1 immune markers upon treatment: CD8α mRNA (6.1-fold; p=0.034), GZMB (3.5-fold; p=0.058), ratios of CD8α/FOXP3 and GZMB/FOXP3 (5.7-fold; p=0.036, and 7.6-fold; p=0.024 respectively), and CTL attractants CXCL10 (2.6-fold; p=0.104) and CCL5 (3.3-fold; p=0.019), successfully meeting the primary endpoint. In contrast, neither Treg marker Foxp3 nor Treg attractants CCL22 or CXCL12 were enhanced. These TME changes were accompanied by transient decreases in circulating CD3+CD8+ CTLs and CD3-CD56+ NK cells (but not Tregs), selectively affecting the cells expressing CXCR3 (receptor for CXCL10), but not CCR4 or CXCR4 (receptors for CCL22 and CXCL12). Three patients had stable disease lasting 2.4, 2.5 and 3.8 months, as of September 1, 2021 cut-off. An additional patient had a partial response (breast auto-amputation) with massive tumor necrosis observed in the post-CKM biopsy. Conclusion: This proof-of-concept study shows that short-term systemic CKM followed by pembrolizumab is safe and selectively enhances local CTL infiltration in the TME, providing rationale for concurrent CKM and PD1 blockade in prospective phase II studies. Citation Format: Shipra Gandhi, Mateusz Opyrchal, Melissa Grimm, Ronald Slomba, Kathleen Kokolus, Sebastiano Battaglia, Kristopher Attwood, Adrienne Groman, Lauren Williams, Mary Lynne Tarquini, Paul Wallace, Kah Teong Soh, Tracey O'Connor, Amy Early, Ellis Levine, Igor Puzanov, Marc Ernstoff, Pawel Kalinski. Systemic rintatolimod and interferon-α2b selectively reprogram local tumor microenvironment in patients with metastatic triple negative breast cancer for enhanced influx of cytotoxic T-lymphocytes but not regulatory T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT145.

A multicenter, open-label, phase 2 study of odetiglucan (IMPRIME PGG) and pembrolizumab in patients with metastatic breast cancer (mBCA) who have progressed through prior hormonal therapy.

TPS1115 Background: Hormone receptor (HR) positive/ human epidermal growth factor receptor 2-negative (Her2-) breast cancer is generally considered ‘immunologically cold’ in comparison to TNBC or Her2+ breast cancer. Keynote-028 results showed a modest ORR of 12% to anti-PD1 antibody, pembrolizumab (PEM) in previously treated HR+/ HER2-/programmed death ligand 1-positive (PD-L1-positive) advanced breast cancer patients (pts). With limited options available, there is significant unmet need to expand clinical benefit from ICI. Odetiglucan, a novel beta glucan, acts as a pathogen-associated molecular pattern (PAMP) that drives a cascade of immune activating events. It repolarizes the immunosuppressive microenvironment, activates the maturation of antigen presenting cells and significantly enhances efficacy of ICI therapy in preclinical tumor models. In a Ph2 trial (IMPRIME 1) of odetiglucan + PEM in 44 pts with heavily pretreated metastatic TNBC, an ORR=15.9%, DCR=54.5%, mDOR=12.7mo, mPFS=2.86 mo, 12 mo OS rate=57.6%, and mOS=16.4 mo were observed. Clinical benefit was particularly evident in a subset of pts, mTNBC “converters” (12/44 pts) who were originally diagnosed with ER/PR+ disease and progressed through endocrine therapies +/- CDK4/6 inhibitors. In these 12 pts, an ORR=50%, DCR=83%, mDOR=11.2, mPFS=5.6 mo, 12-mo OS rate=64.8%, and mOS=17.4 mo were observed. Methods: Odetiglucan + PEM is now being explored in pts with hormone-resistant metastatic breast cancer (MBC). This is a phase 2, Simon’s 2-Stage study of MBC pts who have progressed through prior hormonal therapy with >1 CDK4/6 inhibitor. Pts will receive odetiglucan 4 mg/kg/wk + PEM 200 mg Q3wk. Stage 1 will enroll 23 pts. If >4 pts have an objective response after 12 wks of treatment, the study will proceed to Stage 2 enrolling an additional 24 pts (N=47). Rejection of the null hypothesis requires >10 objective responses. Main eligibility criteria include: MBC having failed prior hormonal therapy with >1 CDK4/6 inhibitor, <2 chemotherapies, serum ABA ≥20 µg/mL, and no prior ICI exposure. Primary endpoint is ORR (RECIST v1.1); secondary endpoints are PFS, OS, DCR, DoR, and safety. Exploratory objectives assess impact of the treatment combination on immune activating events in peripheral blood and tumor biopsies, and correlate tumor microenvironmental changes with clinical benefit Select subpopulations may be explored. Point estimates with 95% confidence intervals (CIs) of ORR and DCR will be computed. Medians, first, and third quartiles with 95% CI will be estimated using Kaplan-Meier method for other secondary endpoints. Safety parameters will be summarized. The trial is sponsored by HiberCell, Inc. in collaboration with Merck & Co. ̃25 US sites will participate. Clinical trial information: NCT05159778.

Phase I study of intratumoral administration of CF33-HNIS-antiPDL1 in patients with metastatic triple negative breast cancer.

e13070 Background: Despite recent FDA approval of immune checkpoint inhibitor pembrolizumab and drug-antibody conjugate in the treatment of metastatic triple negative breast cancer (mTNBC), the overall survival benefit of these therapies remains modest. Oncolytic virotherapy (OV) utilizes genetically modified viruses to infect and kill cancer cells while sparing healthy cells. CF33-hNIS-anti-PD-L1 (CHECKvacc) is a novel chimeric orthopoxvirus with robust anti-cancer activity in TNBC xenografts. Cells infected with CHECKvacc were shown to express functional human sodium-iodide symporter (hNIS) and anti-PD-L1 proteins. hNIS gene transfer allows tracking of virus by 99mTc single-photon emission computed tomography (SPECT). Our preliminary animal studies demonstrated that tumor cells infected with CHECKvacc successfully secrete functional hNIS and anti-PD-L1. CHECKvacc is safe and well-tolerated, and detects and effectively kills TNBC at doses several magnitudes lower than other OVs in xenograft models. Methods: This study is a first-in-human (FIH) phase I, single center, single arm clinical trial evaluating the safety and tolerability of CHECKvacc intratumoral injection in patients with mTNBC. Key eligibility criteria include patients with unresectable or metastatic TNBC; progressed on at least 2 prior chemotherapies including immune checkpoint inhibitor-containing regimen; ECOG 0-1; RECIST 1.1 measurable disease; and at least one tumor amenable to repeated intratumoral injections. Eligible patients receive CHECKvacc intratumorally at one of 8 assigned dose levels (1 × 105 PFU, 3 × 105 PFU, 1 × 106 PFU, 3× 106 PFU, 1 × 107 PFU, 3 × 107 PFU, 1 × 108 PFU, 3 x 108 PFU) on Days 1 and 15 of each 28-day cycle for a total of 3 cycles of treatment. Primary objectives are to evaluate the safety and tolerability of CHECKvacc (DLTs and other toxicities by CTCAE v5.0). Secondary objectives are to determine optimal biological dose (OBD), recommended phase II dose (RP2D), and response rate by RECIST1.1. Results: The first 3 subjects of dose level 1 will be enrolled sequentially for safety monitoring. Once the initial 3 subjects are treated sequentially, the study will follow the Phase I Queue 3+3 (IQ 3+3) design which expands a dose level up to 8 subjects after a single DLT has been observed. Enrollment to the final RP2D may be expanded to include up to 12 patients for efficacy assessment. The estimated targeted accrual is 33 patients (minimum) to 78 patients (maximum). Correlative aims include assessing viral kinetics, viral plaque assay, 99mTc SPECT imaging for virus tracking, peripheral blood and tumor tissue for antiviral immune activation, and tumor microenvironment changes in association with response to therapy. Conclusions: This FIH trial of CF33-hNIS-antiPD-L1 intratumoral injection in patients with mTNBC is currently underway. Clinical trial information: NCT05081492.

Emerging Blood-Based Biomarkers for Predicting Immunotherapy Response in NSCLC.

Simple SummaryTreatment with immunotherapy has been established as a standard treatment for lung cancer in recent years. Unfortunately, still, only a small proportion of patients benefit from the treatment, being the first leading cause of cancer death worldwide. Therefore, there is an urgent need for predictive biomarkers to help clinicians to discern whose patients are more likely to respond to immunotherapy. Since liquid biopsy opens the door to select patients and monitor the response during the treatment in a non-invasive way, in this review, we focus on the most relevant and recent results based on blood soluble biomarkers.Immunotherapy with Immune Checkpoint Inhibitors (ICIs) has demonstrated a profitable performance for Non-Small Cell Lung Cancer (NSCLC) cancer treatment in some patients; however, there is still a percentage of patients in whom immunotherapy does not provide the desired results regarding beneficial outcomes. Therefore, obtaining predictive biomarkers for ICI response will improve the treatment management in clinical practice. In this sense, liquid biopsy appears as a promising method to obtain samples in a minimally invasive and non-biased way. In spite of its evident potential, the use of these circulating biomarkers is still very limited in the real clinical practice, mainly due to the huge heterogeneity among the techniques, the lack of consensus, and the limited number of patients included in these previous studies. In this work, we review the pros and cons of the different proposed biomarkers, such as soluble PD-L1, circulating non-coding RNA, circulating immune cells, peripheral blood cytokines, and ctDNA, obtained from liquid biopsy to predict response to ICI treatment at baseline and to monitor changes in tumor and tumor microenvironment during the course of the treatment in NSCLC patients.

Topical pH Sensing NIR Fluorophores for Intraoperative Imaging and Surgery of Disseminated Ovarian Cancer.

Fluorescence‐guided surgery (FGS) aids surgeons with real‐time visualization of small cancer foci and borders, which improves surgical and prognostic efficacy of cancer. Despite the steady advances in imaging devices, there is a scarcity of fluorophores available to achieve optimal FGS. Here, 1) a pH‐sensitive near‐infrared fluorophore that exhibits rapid signal changes in acidic tumor microenvironments (TME) caused by the attenuation of intramolecular quenching, 2) the inherent targeting for cancer based on chemical structure (structure inherent targeting, SIT), and 3) mitochondrial and lysosomal retention are reported. After topical application of PH08 on peritoneal tumor regions in ovarian cancer‐bearing mice, a rapid fluorescence increase (< 10 min), and extended preservation of signals (> 4 h post‐topical application) are observed, which together allow for the visualization of submillimeter tumors with a high tumor‐to‐background ratio (TBR > 5.0). In addition, PH08 is preferentially transported to cancer cells via organic anion transporter peptides (OATPs) and colocalizes in the mitochondria and lysosomes due to the positive charges, enabling a long retention time during FGS. PH08 not only has a significant impact on surgical and diagnostic applications but also provides an effective and scalable strategy to design therapeutic agents for a wide array of cancers.

The role of PFKFB3 in maintaining colorectal cancer cell proliferation and stemness.

Since generally confronting with the hypoxic and stressful microenvironment, cancer cells alter their glucose metabolism pattern to glycolysis to sustain the continuous proliferation and vigorous biological activities. Bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) isoform 3 (PFKFB3) functions as an effectively modulator of glycolysis and also participates in regulating angiogenesis, cell death and cell stemness. Meanwhile, PFKFB3 is highly expressed in a variety of cancer cells, and can be activated by several regulatory factors, such as hypoxia, inflammation and cellular signals. In colorectal cancer (CRC) cells, PFKFB3 not only has the property of high expression, but also probably relate to inflammation-cancer transformation. Recent studies indicate that PFKFB3 is involved in chemoradiotherapy resistance as well, such as breast cancer, endometrial cancer and CRC. Cancer stem cells (CSCs) are self-renewable cell types that contribute to oncogenesis, metastasis and relapse. Several studies indicate that CSCs utilize glycolysis to fulfill their energetic and biosynthetic demands in order to maintain rapid proliferation and adapt to the tumor microenvironment changes. In addition, elevated PFKFB3 has been reported to correlate with self-renewal and metastatic outgrowth in numerous kinds of CSCs. This review summarizes our current understanding of PFKFB3 roles in modulating cancer metabolism to maintain cell proliferation and stemness, and discusses its feasibility as a potential target for the discovery of antineoplastic agents, especially in CRC.

High expression of RIPK2 is associated with Taxol resistance in serous ovarian cancer

BackgroundTaxol resistance in serous ovarian cancer is responsible for its poor prognosis, yet the underlying mechanism is still poorly understood. Thus, we probed the mechanism of Taxol resistance in serous ovarian cancer with multiple bioinformatic methods to provide novel insights into potential therapies.MethodsThe differentially expressed genes (DEGs) in Taxol-sensitive and Taxol-resistant cell lines and their relationship with the overall survival (OS) and progression-free interval (PFI) of ovarian cancer patients were analyzed using gene expression datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The role of receptor interacting serine/threonine kinase 2 (RIPK2) was validated via identification of its coexpressed genes, functional analysis and generation of a protein-protein interaction (PPI) network. The single sample gene set enrichment analysis (ssGSEA) was used to explore immune infiltration, and genomic alterations of RIPK2 were also analyzed via cBio Cancer Genomics Portal (cBioProtal).ResultsRIPK2 was highly expressed in Taxol resistant ovarian cancer cell lines, and its high expression was also linked with shorter OS and PFI in serous ovarian cancer patients. The PPI network analysis and pathway analysis demonstrated that RIPK2 might participate in the positive regulation of NF-κB transcription factor activity. RIPK2 expression was related to tumor microenvironment alterations, which might participate in the formation of Taxol resistance.ConclusionsOur studies suggested that high expression of RIPK2 is related to Taxol resistance in serous ovarian cancer, and that RIPK2 induces Taxol resistance through NOD1/RIPK2/NF-κB inflammatory pathway activation and tumor microenvironment changes.