Increased Tumor Cell Research Articles

Tumor cell HLA class I expression and pathologic response following neoadjuvant immunotherapy for newly diagnosed head and neck cancer

ObjectivesBiomarkers are needed to identify patients likely to respond to neoadjuvant immunotherapy (NIT) prior to receiving definitive treatment. Materials and MethodsWe hypothesized that expression of tumor cell HLA class I would correlate with pathologic response (PR) following NIT for primary untreated head and neck cancer. Multispectral immunofluorescence of pre- and post-treatment biopsy specimens from a neoadjuvant study of bintrafusp alfa, a dual TGF-β and PD-L1 inhibitor, was performed. ResultsDiscordant expression of tumor cell HLA class I and PD-L1 measured by multispectral immunofluorescence was observed with most positive tumor cells expressing HLA class I or PD-L1 but not both. Spatial analysis revealed colocalization between tumor parenchyma T cells and HLA class I positive tumors cells, but no clear colocalization between T cells and PD-L1 positive tumor cells. Greater pre-treatment tumor cell HLA class I expression, but not PD-L1 expression or tumor T cell infiltration, correlated with the development of a PR. Additionally, increased tumor cell HLA class I expression after NIT compared to before NIT correlated with development of a PR, whereas inconsistent changes in PD-L1 and T cell infiltration were observed after treatment in all patients. ConclusionsThese data provide the rationale for the study of tumor cell HLA class I expression in larger prospective studies powered to determine the performance of biomarkers of PR in newly diagnosed HNSCC patients receiving NIT.

AL008 Enhances Myeloid Antitumor Function by Inhibiting SIRPα Signaling and Activating Fc Receptors.

Antagonizing the CD47-signal regulatory protein (SIRP)α pathway, a critical myeloid checkpoint, promotes antitumor immunity. In this study, we describe the development of AL008, a pan-allelic, SIRPα-specific Ab that triggers the degradation of SIRPα and, concurrently, stimulates FcγR activation of myeloid cells through an engineered Fc domain. AL008 showed superior enhancement of phagocytosis of tumor cells opsonized with antitumor Ag Abs compared with another SIRPα Ab tested. Unlike ligand-blocking SIRPα Abs, AL008 demonstrated single-agent activity by increasing tumor cell engulfment by human monocyte-derived macrophages even in the absence of opsonizing agents. This effect was due to enhanced Fc function, as blocking FcγR2A abrogated AL008-mediated phagocytic activity. AL008 also promoted human monocyte-derived dendritic cell-mediated T cell proliferation. In humanized mouse models, AL008 induced internalization of SIRPα and increased expression of CD86 and HLA-DR on human tumor-associated macrophages, confirming that the mechanism of action is retained invivo. Monotherapy treatment with AL008 significantly reduced tumor growth in humanized mice implanted with human MDA-MB-231 tumor cells. AL008 also significantly potentiated the effects of T cell checkpoint blockade with anti-programmed death ligand-1 in syngeneic tumor models. This dual and specific mechanism of AL008, to our knowledge, provides a novel therapeutic strategy for targeting myeloid cells for immune activation.

Abstract PR016: Carcinoma-associated mesenchymal stem cells promote ovarian cancer metastasis by increasing tumor heterogeneity through direct mitochondrial transfer

Abstract Ovarian cancer is characterized by early, diffuse metastatic spread with most women presenting with widespread abdominal metastasis at the time of diagnosis. Prior work demonstrates carcinoma-associated mesenchymal stem cells (CA-MSCs) enhance ovarian cancer metastasis through a process of direct cellular interaction and formation of heterocellular CA-MSC and tumor cell complexes. Here we demonstrate CA-MSCs enhance metastasis via increasing tumor cell heterogeneity through mitochondrial donation. CA-MSCs directly interact with ovarian cancer cells forming tunneling nanotubules (TNTs). CA-MSCs transfer live mitochondria via these TNTs to ovarian cancer cells. This mitochondrial donation preferentially occurs in ovarian cancer cells with the least endogenous mitochondria (‘mito poor’ cancer cells). Mito poor cancer cells demonstrate decreased proliferation, increased sensitivity to chemotherapy and decreased oxidative phosphorylation compared to ‘mito rich’ cancer cells. CA-MSCs rescue the phenotype of these mito poor cancer cells restoring their proliferative capacity, increasing chemotherapy resistance and increasing oxidative phosphorylation. We validated these findings in a fully autologous system using CA-MSCs and cancer cells derived from the same patient to prevent confounding effects of cellular response to foreign organelle/DNA. Using a knockdown of the mitochondrial motor protein, MIRO1, we demonstrate mitochondrial transfer is necessary for the CA-MSC-mediated rescue of mito poor cancer cells. We developed a haplotype-specific quantification of mitochondrial DNA to differentiate CA-MSC derived mitochondria from endogenous tumor cell mitochondria. We used this system to both quantify the amount of CA-MSC mitochondrial donation and to demonstrate CA-MSC donated mitochondria persist in tumor cells over at least 14 days. Importantly, CA-MSC mitochondrial donation occurs in vivo and is associated with decreased survival in an orthotopic ovarian cancer mouse model. A DNA barcoding system was used to quantify tumor cell clonal heterogeneity. Using this system, we demonstrate CA-MSCs significantly enhance tumor cell heterogeneity, particularly during metastasis. This increase in tumor cell heterogeneity is dependent on CA-MSC mitochondrial transfer. Collectively, we report CA-MSC mitochondrial transfer as a critical mediator of ovarian cancer survival, heterogeneity and metastasis representing a potentially powerful therapeutic target. Citation Format: Leonard G. Frisbie, Catherine A. Pressimone, Huda I. Atiya, Alexander T. Pearson, Lan G. Coffman. Carcinoma-associated mesenchymal stem cells promote ovarian cancer metastasis by increasing tumor heterogeneity through direct mitochondrial transfer [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR016.

Abstract B039: Adipocyte regulation of ER stress and mTOR signaling in bone-metastatic PCa: The role of stearoyl-CoA desaturase

Abstract Bone metastatic disease is correlated with increased morbidity and mortality in prostate cancer (PCa) patients. Current treatments for bone-metastatic PCa are palliative, and new targets for therapy are desperately needed for this presently incurable disease. Various studies have determined that the supportive nature of the bone marrow (BM) niche and crosstalk between the bone-resident cells and the tumor lead to increased tumor cell survival and escape from therapy. Specifically, studies from our lab highlighted the critical role of bone marrow adipose tissue and its expansion due to obesity or age in PCa progression and chemoresistance in bone. Bone marrow adipocytes are metabolically active cells that, through secretion of lipids and adipokines, have the potential to affect the neighboring cells, including the tumor cells that have disseminated into the BM niche. We have shown previously that interaction with marrow adipocytes promotes lipid uptake by PCa cells, modulates their metabolic phenotype, and activates pro-survival signaling and ER/oxidative stress pathways; however, the molecular mechanisms behind the tumor-promoting effects of adipocytes are not understood. The main objective of this study was to investigate the molecular mechanisms underlying lipid-induced stress during tumor cell-adipocyte crosstalk and its functional relationship to tumor growth and progression in bone. Our preliminary data showed that adipocyte-tumor cell crosstalk increases lipid peroxidation in PCa cells, which coincides with augmented expression of ER stress markers and activation of mTOR signaling. We also observed that PCa cells under adipocyte exposure had augmented gene expression of stearoyl-CoA desaturase (SCD), an ER-resident enzyme responsible for the conversion of saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA). We, therefore, hypothesized that SCD is a metabolic regulator that protects metastatic PCa cells against lipid-induced toxicity and ER stress and promotes tumor survival via activation of mTOR signaling. Here, we show that the inhibition of mTORC1 by Everolimus (EVO) decreases the gene expression of ER stress markers in PCa cells exposed to adipocytes. We also demonstrate that adipocyte-mediated induction of ER stress coincides with an increase in active mTOR signaling in PCa cells, indicating possible bidirectional crosstalk between ER stress and mTOR pathways. Notably, inhibition of SCD in PCa cells grown in Transwell co-culture with adipocytes by either siRNA or the small molecule inhibitor CAY10566 leads to reduced mTOR signaling and an increase in lipid peroxidation in PCa cells. SCD inhibition also increases the gene and protein expression of ER stress markers, specifically XBP1 (S) and ATF4, suggesting apoptosis induction. Collectively, our results place SCD as a mediator of ER stress-mTOR crosstalk and a promoter of tumor survival and suggest that targeting SCD activity might be a viable approach to rewire tumor metabolism, and inhibit metastatic progression in bone and sensitize PCa tumors to therapy. Citation Format: Alexis Wilson, Mackenzie Herroon, Shane Mecca, Laimar Garmo, Izabela Podgorski. Adipocyte regulation of ER stress and mTOR signaling in bone-metastatic PCa: The role of stearoyl-CoA desaturase [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B039.

Breast tumor metastasis following filgrastim administration due to the SDF-1/CXCR4 pathway.

Filgrastim, a recombinant type of granulocyte-colony stimulating factor (G-CSF), has a high potential to manage chemotherapy-induced leukopenia. It can increase stromal cell-derived factor 1 (SDF-1) which may stimulate C-X-C chemokine receptor type 4 (CXCR4) to migrate bone marrow-derived stem/progenitor cells to the bloodstream. Here, we aimed to investigate in vitro and in vivo effects of filgrastim on cell migration, invasion, and metastasis. A lentivirus vector of the anti-CXCR4 receptor was first used for the CXCR4 knockout. Effects of filgrastim on cell proliferation and migration were then investigated on 4T1 cells by Transwell migration and wound healing assay. At last, the effects of filgrastim on cell metastasis and the possible involved mechanisms have been investigated in a metastatic murine breast tumor. The knockout of the CXCR4 receptor could lead to a decrease in cell proliferation, migration, and invasion of the 4T1 cells. Filgrastim could directly target SDF-1 and upregulate the expression of the CXCR4 receptor. The knockout of the CXCR4 receptor reduced cell metastasis in an animal model of breast cancer. CXCR4 receptor stimulation by the filgrastim-affected pathways is a conserved evolutionary response that could increase cancer cell proliferation and consequent cell metastasis. Our results suggest that the activation of the CXCR4 receptor is a conserved evolutionary response that can increase cell proliferation, migration, and consequent metastasis. It seems that filgrastim may increase the chance of cancer cell metastasis in people continuously receiving it to increase the number of neutrophils. Filgrastim induces the SDF-1/CXCR4 axis on tumor cell growth. SDF-1 and its receptor CXCR4 are vital targets for filgrastim. The CXCR4 can stimulate the PI3K/AKT, NF-κB, and JAK/STAT signaling pathways. The SDF-1/CXCR4 pathway promotes cell chemotaxis and proliferation via MAPKs signaling. It also enhances cell survival, proliferation, and angiogenesis, increasing tumor cell metastasis. The STAT3-mediated inflammation is essential for tumorigenesis processes, and Akt, Wnt, STAT3, and CXCR4 signaling pathways are all correlated. CXCR4 = C-X-C chemokine receptor type 4, SDF-1 = stromal-derived-factor-1, MAPK = mitogen activated protein kinase; NF-κB = nuclear factor-κB, PI3K = phosphoinositide 3-kinase, JAK = Janus kinase, STAT = signal transducer and activator of transcription, PLC = phospholipase C, PKC = Protein kinase C, GRK = G protein-coupled receptor kinase.

The promise of targeting heme and mitochondrial respiration in normalizing tumor microenvironment and potentiating immunotherapy.

Cancer immunotherapy shows durable treatment responses and therapeutic benefits compared to other cancer treatment modalities, but many cancer patients display primary and acquired resistance to immunotherapeutics. Immunosuppressive tumor microenvironment (TME) is a major barrier to cancer immunotherapy. Notably, cancer cells depend on high mitochondrial bioenergetics accompanied with the supply of heme for their growth, proliferation, progression, and metastasis. This excessive mitochondrial respiration increases tumor cells oxygen consumption, which triggers hypoxia and irregular blood vessels formation in various regions of TME, resulting in an immunosuppressive TME, evasion of anti-tumor immunity, and resistance to immunotherapeutic agents. In this review, we discuss the role of heme, heme catabolism, and mitochondrial respiration on mediating immunosuppressive TME by promoting hypoxia, angiogenesis, and leaky tumor vasculature. Moreover, we discuss the therapeutic prospects of targeting heme and mitochondrial respiration in alleviating tumor hypoxia, normalizing tumor vasculature, and TME to restore anti-tumor immunity and resensitize cancer cells to immunotherapy.

Expression of stathmin in oral squamous cell carcinoma and its correlation with tumour proliferation.

Stathmin is a member of microtubule-associated protein. Inhibition of Stathmin expression can interfere with tumour progression and also alter the sensitivity of tumour cells to microtubule-targeting agents. Thus, it could be a potential therapeutic target for planning new treatment strategies. To study expression of Stathmin in different histological grades of oral squamous cell carcinoma (OSCC) and its correlation with Ki67 index. This study was an observational retrospective and prospective study conducted during a period of two and half years from January 2015 to June 2017 at ESI-PGIMSR Maniktala, Kolkata where 52 cases of OSCC were studied. Haematoxylin and eosin sections were reviewed and representative paraffin blocks were selected. Immunostains were performed using antibody clones for Stathmin and Ki67. For Stathmin scoring, Segersten scoring system was applied. Statistical analysis was done by Graph Pad Prism using Krusher Wallis Test and one-way ANOVA test. Spearman's coefficient was used to establish corelation between Ki 67 and Stathmin overexpression. In this study, it is found that strong Stathmin expression score (4-9) was detected mostly (82.35%) in moderately differentiated (MD) OSCC and poorly differentiated (PD) OSCC (100%), whereas in contrast, 60% of well-differentiated OSCC showed negative-to-weak Stathmin score (1-3). Mean Ki67-labelling index for well-differentiated carcinoma was 32.37%, for moderately differentiated carcinoma was 60.89, and poorly differentiated carcinoma was 86.15%, which demonstrated increased tumour cell proliferation with progression of histological grades of OSCC. Stathmin expression was higher in MD OSCC to PD OSCC compared to well-differentiated carcinoma and its overexpression was significantly correlated with Ki67 index. Thus, Stathmin is overexpressed in higher grades and is correlated with high proliferation of tumour with a potential role as therapeutic target.

Quantification of Radiomics features of Peritumoral Vasogenic Edema extracted from fluid-attenuated inversion recovery images in glioblastoma and isolated brain metastasis, using T1-dynamic contrast-enhanced Perfusion analysis.

The peritumoral vasogenic edema (PVE) in brain tumors exhibits varied characteristics. Brain metastasis (BM) and meningioma barely have tumor cells in PVE, while glioblastoma (GB) show tumor cell infiltration in most subjects. The purpose of this study was to investigate the PVE of these three pathologies using radiomics features in FLAIR images, with the hypothesis that the tumor cells might influence textural variation. Ex vivo experimentation of radiomics analysis of T1-weighted images of the culture medium with and without suspended tumor cells was also attempted to infer the possible influence of increasing tumor cells on radiomics features. This retrospective study involved magnetic resonance (MR) images acquired using a 3.0-T MR machine from 83 patients with 48 GB, 21 BM, and 14 meningioma. The 93 radiomics features were extracted from each subject's PVE mask from three pathologies using T1-dynamic contrast-enhanced MR imaging. Statistically significant (< 0.05, independent samples T-test) features were considered. Features maps were also computed for qualitative investigation. The same was carried out for T1-weighted cell line images but group comparison was carried out using one-way analysis of variance. Further, a random forest (RF)-based machine learning model was designed to classify the PVE of GB and BM. Texture-based variations, especially higher nonuniformity values, were observed in the PVE of GB. No significance was observed between BM and meningioma PVE. In cell line images, the culture medium had higher nonuniformity and was considerably reduced with increasing cell densities in four features. The RF model implemented with highly significant features provided improved area under the curve results. The possible infiltrative tumor cells in the PVE of the GB are likely influencing the texture values and are higher in comparison with BM PVE and may be of value in the differentiation of solitary metastasis from GB. However, the robustness of the features needs to be investigated with a larger cohort and across different scanners in the future.

The development of multifunctional sulfated polyguluronic acid-based polymeric micelles for anticancer drug delivery

Numerous disseminated tumor cells specifically overexpress P-selectin. Therefore, it was thought to be a potential target for tumor therapy. Herein, we described a novel P-selectin-targeted glycosyl ligand-sulfated polyguluronic acid (PGS), as an oriented carrier of P-selectin-targeted drug delivery system. Specifically, the PGS-SS-DOX polymeric micelles were constructed to confirm the practicability of the PGS carrier as a new P-selectin-targeted ligand. PGS-SS-DOX micelles comprised P-selectin-targeted PGS, doxorubicin (DOX) as an anticarcinogen, and pH/redox dual-sensitive bio-linker facilitating drug release in tumor tissues. In vitro and in vivo data showed that PGS-SS-DOX micelles significantly increased tumor cell killing capacity and exhibited a favorable biocompatibility comparison with Free-DOX. This work proved that PGS was an ideal low immunogenic, biodegradable drug carrier for the delivery of anti-cancer drugs. The facile PGS-SS-drug micelle system provided enormous opportunities for treating disseminated tumors utilizing many irreplaceable anticarcinogens.

Lethal effects of mitochondria via microfluidics.

Tumor cells can respond to therapeutic agents by morphologic alternations including formation of tunneling nanotubes. Using tomographic microscope, which can detect the internal structure of cells, we found that mitochondria within breast tumor cells migrate to an adjacent tumor cell through a tunneling nanotube. To investigate the relationship between mitochondria and tunneling nanotubes, mitochondria were passed through a microfluidic device that mimick tunneling nanotubes. Mitochondria, through the microfluidic device, released endonuclease G (Endo G) into adjacent tumor cells, which we referred to herein as unsealed mitochondria. Although unsealed mitochondria did not induce cell death by themselves, they induced apoptosis of tumor cells in response to caspase-3. Importantly, Endo G-depleted mitochondria were ineffective as lethal agents. Moreover, unsealed mitochondria had synergistic apoptotic effects with doxorubicin in further increasing tumor cell death. Thus, we show that the mitochondria of microfluidics can provide novel strategies toward tumor cell death.

Abstract B009: Sub-oxidant ascorbate sensitizes glioblastoma stem cells to DNA damaging agents by inducing DNA de-methylation and altering the chromatin landscape

Abstract Gliomas are the most common primary brain cancer and glioblastoma (GBM) represents the most aggressive and lethal subtype. The high GBM morbidity and mortality are due to profound genetic, epigenetic, and phenotypic heterogeneity within the tumor that contributes to an almost guaranteed development of therapeutic resistance and tumor recurrence. GBM stem-like cells (GSCs) are tumor cell sub-populations that disproportionately drive tumor propagation, therapy resistance, and tumor recurrence. Epigenetic deregulation is thought to play a fundamental role in the formation and expansion of GSCs and their contributions to oncogenesis. However, the epigenetic reprogramming events that contribute to GSC tumor propagation and therapy resistance remain elusive. DNA methylation (5mC) can be modified to generate hydroxymethylation (5hmC) or reversed through enzymatic reactions coordinated by the Ten-Eleven Translocation (TET) enzymes. Multiple studies show a decrease in both 5hmC levels and TET expression in gliomas and a correlation between loss of 5hmC and increasing glioma grade and poor prognosis. These observations suggest that re-establishing TET activity and 5hmC levels in tumor cells will have tumor suppressive effects. Ascorbate (vitamin C) has gained pre-clinical interest as an attractive antitumor agent for GBM due to its ability to regulate redox status, cross the blood-brain barrier, and enhance TET enzymatic activity; therefore, providing an innovative potential epigenetic therapeutic for GBM. We show that low-dose ascorbate (i.e. sub-pro-oxidant concentrations) enhances TET activity and elevates global 5hmC levels concurrent with decreases in 5mC in GSCs. These changes in DNA methylation correlate with a decrease in self-renewal capacity and increased tumor cell death induced by temozolomide (TMZ) and ionizing radiation (IR). We also present a novel mechanism whereby ascorbate increases the histone mark H3K36me3 by upregulating GSC expression of the histone methyltransferase NSD1 via demethylation of the gene promoter region. We propose that the enhanced susceptibility of ascorbate-treated tumor cells to DNA damage therapies results from increased H3K36me3-induced euchromatin states that increases genomic susceptibility to DNA damage. These findings support the need to better understand the epigenetic, transcriptomic, and chromatin architectural effects of ascorbate in cancer. These results also strongly indicate that ascorbate could serve as an adjunct to standard-of-care GBM treatment by restoring TET tumor-suppressor activity and re-sensitizing GSCs to conventional radio- and chemotherapeutics. Citation Format: Maureen Malloy, Sweta Sudhir, Harmon Khela, Maria Fagundo-Lugo, Bachchu Lal, Joseph Bressler, John Laterra, Hernando Lopez Bertoni. Sub-oxidant ascorbate sensitizes glioblastoma stem cells to DNA damaging agents by inducing DNA de-methylation and altering the chromatin landscape. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B009.

Lactate regulators contribute to tumor microenvironment and predict prognosis in lung adenocarcinoma.

Lactic acid, as a product of glycolysis, increases tumor cell migration and the invasion of tumor cells in the tumor microenvironment. Besides this, lactic acid promotes the expression of programmed death-1 expression (PD-1) in regulatory T cells, which could cause the failure of PD-1 blockade therapy. However, the implications of lactic acid in the tumor microenvironment of lung adenocarcinoma (LUAD) remain largely unclear. We performed unsupervised consensus clustering to identify lactic-associated subtypes using expression profile of lactate regulators in LUAD. Differentially expressed genes (DEGs) associated with lactic-associated subtypes was used to construct lactate signature (LaSig) using LASSO regression algorithm. Immune infiltration analysis was conducted by ESTIMATER and drug sensitivity was estimated by R package called "pRRophetic". The difference between two groups was calculated using Wilcox rank sum test and correlation analysis was calculated using Pearson correlation coefficient. In this study, we evaluated DNA methylation and the mutation frequency of lactate regulators and found lactate regulators showed low mutation frequency in the TCGA-LUAD cohort, except TP53. At the RNA level, the expression level of lactate regulators was significantly associated with the immune cell component. In particular, expression of LDHA was positively correlated with CD4 T cell, CD8 T cell, M1 macrophages, and the enrichment score of multiple immune pathways. Two clusters were defined using the gene expression level of lactate regulators, and LDHA was significantly upregulated in cluster 1 with poor overall survival. A lactate signature (LaSig) had a robust performance in predicting the survival rate and immunotherapy response of LUAD patients. Moreover, patients in the high LaSig group may be more likely to benefit from these drugs (Cisplatin, Erlotinib, Gemcitabine, and Vinblastine) than those in the low LaSig group. In summary, our study explores the role of lactate regulators in guiding the clinical treatment of lung adenocarcinoma and provides additional help to supplement traditional molecular subtypes.

Phase 1b/2 Study of Autologous CD30.CAR-T Cells in Combination with Nivolumab in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma after Failure of Frontline Therapy (ACTION)

Background Autologous CD30-directed chimeric antigen receptor T-cell (CD30.CAR-T) therapy is well tolerated, with significant clinical efficacy in relapsed or refractory (r/r) classical Hodgkin Lymphoma (cHL) patients, as demonstrated in phase 1/2 clinical studies [LCCC 1532-ATL (NCT02690545) and H-37966 (RELY-30, NCT02917083); Ramos et al., 2020] and the pilot part of CHARIOT phase 2 trial (TESSCAR001, NCT0535288; Ahmed et al., 2021). cHL is characterized by the presence of malignant Hodgkin Reed-Sternberg (HRS) cells, which frequently harbor amplifications in chromosome 9p24.1, leading to overexpression of programmed death-ligand 1 (PD-L1) (Chen et al., 2017). The efficacy and safety profile of programmed death (PD)-1 checkpoint inhibitors, nivolumab and pembrolizumab, in cHL have been well demonstrated (Chen et al., 2017; Kuruvilla et al., 2021; Younes et al., 2016), with FDA approval for r/r cHL. PD-1 checkpoint inhibitors, in combination with CD30-directed antibody therapy (brentuximab vedotin) or other chemotherapies, have also shown high efficacy in this setting (Advani et al., 2021; Mei et al., 2022; Moskowitz et al., 2021). Preclinical studies demonstrated potential synergism between PD-1 checkpoint inhibitors and CAR-T cell therapy, where PD-1 or PD-L1 antibodies boost CAR-T cell therapy and promote increased tumor cell death in vivo (Cherkassky et al., 2016; Gargett et al., 2016; Moon et al., 2014). Despite multiple treatment options available in the second line therapy for cHL, 10-40% of patients do not achieve response to salvage chemotherapy (von Keudell and Younes, 2019). Additionally, salvage chemotherapies are associated with short-term toxicity, long-term morbidity and non-lymphoma-related mortality (de Vries et al., 2021; Dores et al., 2020). We have designed a phase 1b/2 trial (ACTION) to evaluate the safety and antitumor activity of CD30.CAR-T cells combined with nivolumab in advanced cHL patients in whom standard frontline chemotherapy has failed. Study Design and Methods Approximately 15 adult and pediatric patients (≥12 years old) with r/r cHL following progressive disease after standard frontline therapy will be enrolled in the present single-arm, phase 1b/2, multicenter study (ClinicalTrials.gov NCT05352828). Following a leukapheresis procedure for manufacture of CD30.CAR-T cells, patients will be treated with 4 cycles of nivolumab every 4 weeks (Q4W), and a single infusion of CD30.CAR-T cells (given between nivolumab Cycles 2 and 3, and after lymphodepletion with bendamustine and fludarabine). At the end of treatment (EOT) visit (4 weeks after nivolumab Cycle 4) and at the Investigators' discretion, patients with complete response (CR), partial response (PR), or stable disease (SD) will undergo either standard treatment with autologous stem cell transplant (ASCT group) or continue nivolumab Q4W for up to 6 additional cycles, unless patients experience either progressive disease (PD) or unacceptable toxicity, whichever occurs earlier (non-ASCT group). Patients will be followed with periodic response assessments and safety monitoring until end of study (EOS), approximately 3 years after leukapheresis. Long-term follow-up will continue for up to 15 years. The primary objective of this study is to assess the safety of autologous CD30.CAR-T in combination with nivolumab. The key secondary objective is to evaluate potential anti-tumor activity, as assessed by the CR rate of autologous CD30.CAR-T in combination with nivolumab at EOT in 14 evaluable patients, as per Lugano Classification Revised Response System for malignant lymphoma (Cheson et al., 2014). Other secondary objectives are to assess the overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS) of patients in the ASCT and non-ASCT groups. This study is ongoing and one patient has been enrolled to date. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Biological impact and therapeutic perspective of targeting PI3K/Akt signaling in hepatocellular carcinoma: Promises and Challenges

Cancer progression results from activation of various signaling networks. Among these, PI3K/Akt signaling contributes to proliferation, invasion, and inhibition of apoptosis. Hepatocellular carcinoma (HCC) is a primary liver cancer with high incidence rate, especially in regions with high prevalence of viral hepatitis infection. Autoimmune disorders, diabetes mellitus, obesity, alcohol consumption, and inflammation can also lead to initiation and development of HCC. The treatment of HCC depends on the identification of oncogenic factors that lead tumor cells to develop resistance to therapy. The present review article focuses on the role of PI3K/Akt signaling in HCC progression. Activation of PI3K/Akt signaling promotes glucose uptake, favors glycolysis and increases tumor cell proliferation. It inhibits both apoptosis and autophagy while promoting HCC cell survival. PI3K/Akt stimulates epithelial-to-mesenchymal transition (EMT) and increases matrix-metalloproteinase (MMP) expression during HCC metastasis. In addition to increasing colony formation capacity and facilitating the spread of tumor cells, PI3K/Akt signaling stimulates angiogenesis. Therefore, silencing PI3K/Akt signaling prevents aggressive HCC cell behavior. Activation of PI3K/Akt signaling can confer drug resistance, particularly to sorafenib, and decreases the radio-sensitivity of HCC cells. Anti-cancer agents, like phytochemicals and small molecules can suppress PI3K/Akt signaling by limiting HCC progression. Being upregulated in tumor tissues and clinical samples, PI3K/Akt can also be used as a biomarker to predict patients’ response to therapy.

ANGI-07. THE DUAL ROLE CD97 PLAYS IN GLIOBLASTOMA INVASION AND PROLIFERATION

Abstract INTRODUCTION Increased levels of adhesion G Protein-Coupled Receptor(GPCR) CD97 have been detected in multiple metastatic cancers. Glioblastoma(GBM), also overexpress this receptor, and its levels positively correlate with more aggressive GBM invasion. Here, using primary human GBM cells we investigated the effects of CD97 knockdown and overexpression in-vitro and in-vivo. METHODS CD97 consists of two distinct domains: the large extracellular fragment interacting with ECM and other cell surface receptors and the GPCR core that activates heterotrimeric G Proteins G12/13. To investigate the mechanism of action of CD97, we generated a CD97 knockdown (shCD97) and overexpression (CD97-WT) constructs. Additionally, we generated two functional mutants: a constitutively active mutant lacking the N-Terminus (dNTF) and dominant-negative mutant (H436A) that cannot activate G-Proteins downstream. Glioma Stem Cells(GSC) were transduced by control and CD97 lentiviral constructs and implanted into mouse brains. Once tumors formed the brains were harvested, sliced, and immunostained to evaluate invasion and proliferation. Comparing mutants to control, dunnett’s multiple comparisons test was used for statistical analysis. RESULTS Similar to CD97-WT, we found that both dNTF and H436A, increase tumor volume(p&amp;lt; 0.05). In contrast, the shCD97 mutant decreased tumor size(p&amp;lt; 0.05). Interestingly, only dNTF significantly increased tumor cell proliferation(p&amp;lt; 0.005), yet CD97-WT and H436A affected proliferation of non-tumor cells in the tumor bulk, indicating that this process requires the N-Terminus Fragment. In addition, over-expression of CD97-WT dramatically increases tumor invasion along myelinated fibers of corpus collosum, and tumor spread in the brain tissue(p&amp;lt; 0.05). Yet, the H436A mutant produced large, compact, and non-invasive tumors with sharp borders(p&amp;lt; 0.05) indicating that cell migration requires activation of G12/13. Surprisingly, immunostaining revealed that CD97 knockdown cells displayed invasive phenotype. CONCLUSIONS Our results demonstrate the dual role CD97 plays in GBM invasion and proliferation, alluding to the molecular mechanisms that can potentially be targeted in GBM treatment.

TMIC-04. TARGETING AN ECM PROTEIN INVOLVED IN BREAST CANCER BRAIN METASTASES WITH AN ONCOLYTIC HSV-1

Abstract The beta-1 subunit of integrins (CD29) is important for cell-extracellular matrix interactions. CD29 expression is increased in breast cancer cells, and it promotes their invasion, migration, and intravasation, all of which can result in breast cancer brain metastasis (BCBM). While CD29 expression can be correlated to poor prognosis of breast cancer patients, the median survival of BCBM patients predicted to have better prognosis is 36 months. Despite current BCBM treatments such as craniotomy, reaching the metastatic site is often challenging. Modified viral vectors, such as oncolytic herpes simplex virus (oHSV), have been clinically approved for the treatment of malignant melanoma and brain tumors, as they infect and selectively kill tumor cells. Previously, we identified that combination of oHSV and humanized CD29 blocking antibody (OS2966, aCD29) enhanced viral replication by reducing antiviral immune response, increased tumor cell killing, and rendered survival benefit to tumor bearing mice with intra-tumoral, but not systemic, oHSV administration. These findings suggested that the antitumor efficacy of the therapeutic combination required intratumoral delivery of anti-CD29, since the antibody could not penetrate the blood brain barrier and reach intracranial tumors. To overcome this, we report the generation of an oHSV encoding the scFv-Fc fragment of this antibody (HSV-aCD29), which retains its oncolytic function, and evaluate the impact of HSV-aCD29 on the CD29 downstream signaling. We will further characterize the safety, antitumor efficacy, and assess the immune response to HSV-aCD29 in vitro and in vivo. We anticipate that compared to our control virus, HSV-aCD29 will block CD29 activity which will reduce antiviral immune responses and enhance tumor cell lysis, thus improving oHSV efficacy. Together, this study proposes the blockade of CD29 with the targeted delivery of a CD29 blocking antibody mediated by a novel oHSV as a potential synergistic therapy against BCBM.

Engulfment and Cell Motility 1 (ELMO1) Regulates Tumor Cell Behavior and Predicts Prognosis in Colorectal Cancer.

Engulfment and cell motility 1 (ELMO1) plays a crucial role in the process of migration, chemotaxis, and metastasis of tumor cells. ELMO1 has been implicated in the pathogenesis of various cancers. However, the distinct function of ELMO1 in colorectal cancer (CRC) is unclear. We determined whether ELMO1 affects the oncogenic behavior of CRC cells and investigated its prognostic value in CRC patients. We investigated the impact of ELMO1 on tumor cell behavior using small interference RNA (siRNA) in CRC cell lines, including SW480 and DLD1. The expression of ELMO1 was investigated by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) in cancer tissues and sera obtained from CRC patients. ELMO1 knockdown suppressed tumor cell proliferation in SW480 and DLD1 cells. ELMO1 knockdown-induced apoptosis through up-regulation of caspase-3, -7, and PARP activities and down-regulation of the anti-apoptotic Mcl-1 protein. ELMO1 knockdown-induced cell-cycle arrest by decreasing cyclin D1, cyclin-dependent kinase 2, 4 and 6, and the 25C cell division cycle (CDC25C). ELMO1 knockdown suppressed tumor cell invasion and migration. The expression of E-cadherin was increased, while that of Vimentin and Claudin 1 decreased following ELMO1 knockdown. The phosphorylation levels of PDK1, Akt, and GSK-3β and were down-regulated after ELMO1 knockdown. The expression of ELMO1 was found up-regulated in cancer tissues and sera taken from CRC patients. ELMO1 expression was significantly associated with tumor stage, lymph node metastasis, distant metastases, and poor survival. ELMO1 mediates tumor progression by increasing tumor cell motility and inhibiting apoptosis in human CRC.

Dosimetric Evaluation of Physical Radiation Delivery Limits of a Weak Magnetic Field Generator on GBM Dose Distributions

<h3>Purpose/Objective(s)</h3> To demonstrate the feasibility of meeting dose prescription criteria when using a weak magnetic field generator to support glioblastoma multiforme (GBM) radiation therapy. <h3>Materials/Methods</h3> Weak magnetic fields extend free radical life, and recent experimental results have shown that adding weak magnetic fields (20G-40G) to radiation therapy delivery enhances free radical production, increases tumor cell death, and slows tumor growth. Based on these results, we have built the first clinical unit, consisting of two 63 cm ID Helmholtz coils, spaced 35 cm apart. The coil system is placed such that the patient and Linac couch are within the coils, and the Linac isocenter is coincident with the center of the coil system. We propose to use GBM for our first clinical trial. The limitation imposed by the coil system is a fixed Linac couch angle, constraining the gantry rotation to slightly more than 180°, from lateral to lateral. The impact on treatment plan quality of the corresponding reduction in available beam angles was unknown, so we conducted a treatment planning study to determine if we could provide clinically equivalent treatment plans with the coil in place. We evaluated the treatment plans for 10 GBM patients that had been treated with arc therapy. The prescription was 60 Gy (2 Gy x 30 fractions) for 6 patients, 55.8 Gy (1.8 Gy x 31 fractions) for 2 patients, 54 Gy (1.8 Gy x 30 fractions) for 1 patient, and 40.05 Gy (2.67 Gy x 15 fractions) for 1 patient. The dose was delivered to 95% of the planning target volume (PTV). The PTV margin was 1.3-2 cm surrounding the gross tumor volume. Critical structures, such as the brainstem, optic nerves, and optic chiasm, were contoured and assigned constraints of 62 Gy, 54 Gy, and 54 Gy, respectively. We repeated the treatment planning session using only coplanar arcs that spanned from 90° to 270° (half-arcs). <h3>Results</h3> All the treatment plans generated with the half-arcs met the clinical criteria. The minimum PTV doses for the half arc and clinical plans were 50.0 ± 6.1 Gy and 51.4 ± 7.1 Gy, and the maximum PTV doses were 61.9 ± 7.1 Gy and 62.7 ± 8.6 Gy, respectively. Table 1 summarizes the results of four reported critical structures. All the critical structures met the criteria, and the half-arc plans were neither better nor worse than the clinical plans. <h3>Conclusion</h3> The physical limits imposed by the magnetic field generator on the Linac beam angles do not degrade treatment planning for GBM radiation therapy. The prescribed dose can be adequately delivered while avoiding critical structures. With this groundwork, we can develop clinical trials to evaluate the impact of utilizing weak magnetic fields in radiation therapy.

Germline gain-of-function MMP11 variant results in an aggressive form of colorectal cancer.

Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and nonmatrix proteins. Although most MMPs are secreted as inactive proenzymes and are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11's protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy.

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.