Quiescent Tumor Research Articles

Tumor-Infiltrating Lymphocytes Assessed Using the International TILs Working Group System Are Not Prognostic in Medullary Thyroid Cancer.

Background: Tumor-infiltrating lymphocytes (TILs) are a protective prognostic factor in several solid tumors and predict response to immune checkpoint inhibitor therapy. The prognostic impact of TILs in medullary thyroid cancer (MTC) is poorly understood. Materials and Methods: In this retrospective cohort study, we assessed the TILs profile of primary MTC tumors using the International TILs Working Group system and correlated this with clinicopathological prognostic variables, including the International Medullary Thyroid Cancer Grading System (IMTCGS) grade and survival outcomes. Results: We identified 71 patients with primary MTC tumors who were treated surgically between 1995 and 2016 at the Royal North Shore Hospital in Sydney, Australia. The median (interquartile range) duration of follow-up was 69 (90) months. Using the ITWG system, all patients with MTC had low TILs, with a median (range) of 3% (0-10%). This group was further subdivided into "very low" (0-4%) and "low" (5-10%), and on Cox regression analysis, increasing TILs were associated with increased local recurrence (log-rank p = 0.022, odds ratio [OR] 1.94 [confidence interval or CI 0.61-6.16], p = 0.26), reduced disease-specific survival (log-rank p = 0.015, OR 5.11 [CI 1.01-26.0], p = 0.049), and a trend to decreased distant metastasis-free survival (log-rank p = 0.14). When examining the association between TILs and other prognostic factors, only "high IMTCGS grade" was significantly associated with increased TILs (OR 7.29 [CI 1.21-43.90], p = 0.015). In the multivariable logistic regression analysis, there was no significant association between TILs and local recurrence or disease-specific survival. Conclusions: In our study, the prognostic value of TILs in MTC was limited. Even high-grade MTC can be considered an immune quiescent tumor, and the adverse prognostic factors associated with higher grade tumors outweigh the marginal increase in immune recognition associated with a slight increase in TILs. The low level of TILs in MTC and their lack of correlation with survival suggest that immune checkpoint inhibitor therapy may not be effective.

Targeting Dormant Disseminated Tumor Cells and their Permissive Niche by Pro-Resolving Mediators Derived from Resolution-Phase Macrophages.

Metastatic breast cancer (BC) can recur years after initial treatments and arise from quiescent disseminated tumor cells (QDTC) that resist conventional therapies. To date there are no treatments to target QDTCs. Previously, the fibrotic-like niche (FLN) enriched with Type I collagen (Col-I) was shown to be required for the switch of QDTC to overt metastases. Here, we examined whether artificially reinstating resolution of inflammation, by using soluble mediators secreted by ex-vivo generated pro-resolving macrophages (CM-Mres), will prevent FLN establishment and in turn hinder QDTC outgrowth. Our findings indicate that CM-Mres promoted immune silencing at the metastatic site as part of the resolution process and inhibited the FLN resulting in the inhibition of the metastatic outgrowth in vitro and in vivo. This was due to inhibition of fibroblasts to myofibroblasts differentiation independent of TGFβ1 canonical signaling and the abolishment of Col-I expression. Furthermore, CM-Mres eliminated myofibroblasts as part of the resolution process by inducing an increase in reactive oxygen species (ROS) via NADPH oxidase leading to DNA damage and apoptosis. Moreover, ROS-mediated apoptosis was also induced by CM-Mres in the dormant and outgrowing DTCs. Overall, our findings suggest for the first time that pro-resolving mediators can target both QDTCs and their permissive niche thus preventing BC from recurring. SIGNIFICANCE: Since conventional therapies fail to eradicate QDTCs. Future identification of the pro-resolving mediators secreted by pro-resolving macrophages may serve as a basis for novel therapeutic strategies targeting QDTCs and their metastatic niche.

EPCO-02. HIGH CELLULAR PLASTICITY STATE OF HUMAN MEDULLOBLASTOMA LOCAL RECURRENCE AND DISTANT DISSEMINATION

Abstract Medulloblastoma (MB) is a heterogeneous pediatric brain tumor with variable clinical outcomes. Since current treatments facing limitations due to tumor resistance, less than 10% of relapsed patients survive beyond five years, with poorly understood recurrence mechanisms. To obtain cellular diversity and genetic characters in primary and recurrent MB, we conducted comprehensive analyses utilizing the single-cell/nucleus RNA sequencing (sc/snRNA-seq), snATAC-seq and spatial transcriptomics, complemented with bulk RNA sequencing and matched primary-recurrent whole exon profiles. SHH and Group_3 subgroups revealed distinct cellular subsets, including cycling, differentiated and quiescent tumor cells. Recurrent or disseminated MB displayed a dramatically different cellular ecosystem compared to primary tumors, with varying proportions of shared cell types. Local recurrence exhibited a higher enrichment of cycling tumor cells, while differentiated subset was notably present in disseminated lesions, indicating that local recurrences harbored the stronger proliferative capacity, whereas disseminations relatively well differentiated. Additionally, chromosomal alterations were assessed, reflecting distinct genetic subclones, such as chr7q gain and chr11 loss in Group_3 disseminations. Notably, the emergence of a subpopulation termed “high cellular plasticity (HCP)” during MB progression was noted, characterized by dynamic adaptability and stemness properties. As validated, HCP state was associated with increased chromatin accessibility, contributing to tumor recurrence and immune microenvironment remodeling. Functionally, inhibiting markers associated with HCP cells, such as PTPRZ1, efficiently suppressed MB metastasis in preclinical models. In conclusion, our findings fill the critical knowledge gaps in understanding the cellular diversity, chromosomal alterations and biological dynamics in MB recurrence and dissemination, offering potential therapeutic interventions.

Uveal melanoma immunogenomics predict immunotherapy resistance and susceptibility

Immune checkpoint inhibition has shown success in treating metastatic cutaneous melanoma but has limited efficacy against metastatic uveal melanoma, a rare variant arising from the immune privileged eye. To better understand this resistance, we comprehensively profile 100 human uveal melanoma metastases using clinicogenomics, transcriptomics, and tumor infiltrating lymphocyte potency assessment. We find that over half of these metastases harbor tumor infiltrating lymphocytes with potent autologous tumor specificity, despite low mutational burden and resistance to prior immunotherapies. However, we observe strikingly low intratumoral T cell receptor clonality within the tumor microenvironment even after prior immunotherapies. To harness these quiescent tumor infiltrating lymphocytes, we develop a transcriptomic biomarker to enable in vivo identification and ex vivo liberation to counter their growth suppression. Finally, we demonstrate that adoptive transfer of these transcriptomically selected tumor infiltrating lymphocytes can promote tumor immunity in patients with metastatic uveal melanoma when other immunotherapies are incapable.

Abstract LB314: Organ-specific roles of CD83+ macrophages in breast cancer

Abstract The purpose of this study is to understand how heterogeneity in organ-specific immune infiltrates can lead to Triple Negative Breast Cancer (TNBC) progression and immunotherapies resistance in metastasis. Both innate and adaptive immune responses are essential for breast cancer progression. Using single-cell multiplexing technology on human and murine models of primary and metastatic breast cancers, we investigated the pattern of immune cell infiltration in bone and lung metastases in a temporal manner. The parallel profiling of metastasis sites revealed a strong involvement of the innate immunity in restraining tumor progression with an acute increase in Mrc1+ Cd83+ tumor-associated macrophages at the early stage of lung colonization. Interestingly, the increase was modest in bone metastasis and remained consistent overtime despite bone metastasis progression. In contrast, we saw an increase in the number of Ly6G+ Lrg1+ granulocytes. Profiling of paired TNBC patient samples from the primary and metastatic sites have shown an immunologically quiescent tumor microenvironment in metastasis as compared to the primary tumors. Hence, to further explore the role of Mrc1+ Cd83+ macrophages on therapeutic resistance, we profiled the orthotopic claudin-low TNBC models, T11 and T12, following their exposure to chemotherapy. While responsive tumors displayed a decreased infiltration of Mrc1+ Cd83+ macrophages, the opposite phenotype was observed in non-responders, suggesting their involvement in tumor progression. Overall, our results suggest differential responses in untreated lung/bone metastases and primary tumors indicating divergent roles for Cd83+ TAMs in restraining or promoting tumor progression, while also indicating some crosstalk between neutrophils and stromal cells that aid in bone metastatic progression. These site-specific differences in immune cell phenotype indicate that there is a great need to thoroughly characterize the tumor immune compartment in both the primary and the metastatic setting to improve treatment responses. Considering the inhibitory effect of macrophages on T cells in these models, this study highlights a tissue-specific reprogramming of immune cells in breast cancer metastasis. Citation Format: Swarnima Singh, Igor Bado. Organ-specific roles of CD83+ macrophages in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB314.

Abstract 704: Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance

Abstract Tumor cells undergo biological adaptations in order to survive stress conditions, such as hypoxia and nutrient deprivation. These biological adaptations include the activation of hypoxic survival pathways that play an important role in acquiring radioresistance which becomes most challenging with treatment regimens using high doses of radiation/fraction (e.g., hypofractionated radiotherapy). Therefore, inhibiting key regulators within these pathways and thereby exploiting these survival mechanisms, gives rise to new potential drug targets for combined treatment modalities. This novel strategy investigated in our laboratory relies on the concept of biological cooperation: while ionizing radiation kills primarily well-oxygenated cells, such inhibitors of survival kinases will drive quiescent tumor cells under hypoxia and/or nutrient deprivation either directly into cell death or into re-entry into a proliferative and thereby more ionizing radiation-sensitive state. One of the survival kinases we are investigating in combination with ionizing radiation (IR) is DYRK1B (dual-specificity tyrosine regulated kinase 1B). We demonstrated that the expression of DYRK1B was upregulated under serum-starvation and hypoxia, but not in response to IR. The small molecule DYRK1B inhibitor AZ191 and shRNA-mediated DYRK1B knockdown significantly reduced proliferative activity and clonogenicity of SW620 tumor cells alone and in combination with IR under serum-starved conditions, which correlated with increased ROS levels and DNA damage. Furthermore, AZ191 successfully targeted the hypoxic core of tumor spheroids while IR preferentially targeted normoxic cells in the rim of the spheroids. A combined treatment effect was also observed in patient-derived colorectal carcinoma organoids but not in healthy tissue organoids. This data shows that inhibition of DYRK1B in quiescent tumor cells could drive tumor cells on their own into crisis or into a more radiosensitive cell cycle phase and thus supports our strategy of biological cooperation. In the continuation of this project, we have performed a drug-screen with approx. 3’000 clinically relevant compounds in colorectal tumor cells lines under normoxic and hypoxic conditions in order to explore additional hypoxia-mediated survival pathways contributing to radioresistance. Thereby we aim to identify novel combined treatment modalities to overcome radiotherapy-induced hypoxia and hypoxia-related resistance mechanisms. Citation Format: Claire Beckers, Lazaros Vasilikos, Alba Sanchez-Fernandez, Lorena Moor, Martin Pruschy. Targeting hypoxic survival pathways to overcome radiotherapy-related treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 704.

Abstract A074: Targeting dormant disseminated tumor cells and their permissive niche by pro-resolving mediators derived from resolution-phase macrophages

Abstract Metastatic breast cancer can recur years after primary tumor resection and adjuvant therapy and appears to arise from quiescent disseminated tumor cells (QDTC) that persist at distant sites and resist conventional therapies. To date there are no treatments to target QDTCs. Previously, the fibrotic-like microenvironment enriched with Type I collagen (Col-I) was shown to be required for the switch of QDTC to metastatic outgrowth in lungs of mice. Here, we examined whether soluble mediators secreted by ex-vivo generated pro-resolving CD11blow macrophages (CM-Mres) will reinstate resolution of inflammation thus inhibiting the establishment of the fibrotic-like-niche and in turn prevent the emergence of QDTC to metastatic outgrowth. Our findings indicate that CM-Mres promoted immune silencing of alveolar macrophages at the metastatic site where dormant DTCs reside. Immune silencing is one of the hallmarks of resolution of inflammation. Furthermore, CM-Mres inhibited the establishment of a fibrotic-like niche resulting in the inhibition of the metastatic outgrowth in vitro and in vivo. This was due to inhibition of fibroblasts differentiation to myofibroblasts independent of TGFbeta1 canonical signaling and abolishment of Col-I expression. Furthermore, CM-Mres deactivated myofibroblasts as part of the resolution process by inducing an increase in reactive oxygen species (ROS) via activation of NADPH oxidase. This in turn induced DNA damage leading to Go/G1 arrest and intrinsic apoptosis. Similarly, apoptosis mediated by oxidative stress was induced by CM-Mres in dormant and outbreaking QDTCs. Taken together our results demonstrate for the first time that by reinstating resolution of inflammation via physiological mediators, secreted by pro-resolving macrophages, can prevent metastatic outgrowth of QDTCs at their permissive site. Since conventional therapies fail to eradicate dormant DTCs and their transition to clinical metastasis, our findings hold great promise in the quest to identify novel strategies to prevent or treat recurrent metastatic disease. Citation Format: Odelya Gilon, Keren Weidenfeld, Hadell Samara, Yonatan Feuermann, Sagie Schif-Zuck, Sergei Butenko, Edmond Sabo, Amiram Ariel, Dalit Barkan. Targeting dormant disseminated tumor cells and their permissive niche by pro-resolving mediators derived from resolution-phase macrophages [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A074.

Epigenetic modulation of antitumor immunity and immunotherapy response in breast cancer: biological mechanisms and clinical implications.

Breast cancer (BC) is the most common non-skin cancer and the second leading cause of cancer death in American women. The initiation and progression of BC can proceed through the accumulation of genetic and epigenetic changes that allow transformed cells to escape the normal cell cycle checkpoint control. Unlike nucleotide mutations, epigenetic changes such as DNA methylation, histone posttranslational modifications (PTMs), nucleosome remodeling and non-coding RNAs are generally reversible and therefore potentially responsive to pharmacological intervention. Epigenetic dysregulations are critical mechanisms for impaired antitumor immunity, evasion of immune surveillance, and resistance to immunotherapy. Compared to highly immunogenic tumor types, such as melanoma or lung cancer, breast cancer has been viewed as an immunologically quiescent tumor which displays a relatively low population of tumor-infiltrating lymphocytes (TIL), low tumor mutational burden (TMB) and modest response rates to immune checkpoint inhibitors (ICI). Emerging evidence suggests that agents targeting aberrant epigenetic modifiers may augment host antitumor immunity in BC via several interrelated mechanisms such as enhancing tumor antigen presentation, activation of cytotoxic T cells, inhibition of immunosuppressive cells, boosting response to ICI, and induction of immunogenic cell death (ICD). These discoveries have established a highly promising basis for using combinatorial approaches of epigenetic drugs with immunotherapy as an innovative paradigm to improve outcomes of BC patients. In this review, we summarize the current understanding of how epigenetic processes regulate immune cell function and antitumor immunogenicity in the context of the breast tumor microenvironment. Moreover, we discuss the therapeutic potential and latest clinical trials of the combination of immune checkpoint blockers with epigenetic agents in breast cancer.

The emerging role of osteoclasts in the treatment of bone metastases: rationale and recent clinical evidence.

The occurrence of bone metastasis is a grave medical concern that substantially impacts the quality of life in patients with cancer. The precise mechanisms underlying bone metastasis remain unclear despite extensive research efforts, and efficacious therapeutic interventions are currently lacking. The ability of osteoclasts to degrade the bone matrix makes them a crucial factor in the development of bone metastasis. Osteoclasts are implicated in several aspects of bone metastasis, encompassing the formation of premetastatic microenvironment, suppression of the immune system, and reactivation of quiescent tumor cells. Contemporary clinical interventions targeting osteoclasts have proven effective in mitigating bone-related symptoms in patients with cancer. This review comprehensively analyzes the mechanistic involvement of osteoclasts in bone metastasis, delineates potential therapeutic targets associated with osteoclasts, and explores clinical evidence regarding interventions targeting osteoclasts.

PATH-10. SURGICALLY RESECTED RECURRENT DIFFUSE GLIOMAS: HISTOMOLECULAR SPECTRUM

Abstract Histological treatment-related changes (TRC) may mimic radiological progression in diffuse-gliomas on follow-up after complete therapy. If scans suggest recurrence, patients undergo re-surgery& re-RT, if recurrence is histologically confirmed. We evaluated 1) baseline characteristics of surgically-resected gliomas re-operated for clinicoradiological suspected recurrence; 2) compared histological changes from initial surgery. All adult-type diffuse-gliomas (n=122) re-operated following complete therapy, with available initial& subsequent slides reviewed, classified as per current WHO classification. Clinico-pathological features noted. RESULTS: Baseline median age:36years (IQR:29-44years), M:F=2.21, tumor-bed recurrence in majority (116/122). Of 92 astrocytomas, 47 were IDH-mutant (grade-2:18, grade-3:14, grade-4:15); 31 were IDH-WT (histological grade-2:4, grade-3:5, grade-4:22), IDH not-available (NA) in 14. Among IDH-mutant, 26 were MGMT-methylated (MGMT-M), 11 unmethylated (MGMT-UM). In IDH-WT 4 were MGMT-M, 23 MGMT-UM. Of 30 oligodendrogliomas, 21 were grade-2, 9 grade-3. All grade-2 gliomas (25astrocytoma, 21oligodendroglioma) showed high-grade transformation at re-surgery, with 40% astrocytoma upgrading to grade-3; 60% to grade-4. Six grade-3 astrocytoma recurred at same grade, while 18 as grade-4. Of 43 grade-4, on re-surgery 11 showed predominantly TRC ( >30%) with quiescent residual tumor (4 IDH-mutant, 5 IDH-WT, 2NA), while 32 showed recurrence (10 IDH-mutant, 18 IDH-WT, 4NA), of which 11 had scant (<5%) to focal (6-30%) TRC along-with actively proliferating tumor. Only 2 oligodendrogliomas showed TRC. Median disease-free interval (DFI) between both surgeries was significantly higher for oligodendroglioma (6.76years) than astrocytoma (3.59years, p-value< 0.001); for grade-2 (5.37years)& grade-3 astrocytoma (5.19years) than grade-4 (2.85years, p-value< 0.001); grade-2 versus grade-3 oligodendroglioma (7.02years versus 4.91years, p-value 0.041), &for MGMT-M versus MGMT-UM (5.17 versus 3.02years, p-value< 0.01). Significantly, median DFI was lower for grade-4 astrocytoma with TRC than without (2.34 versus 3.27years, p-value 0.015). CONCLUSIONS: Pseudo-progression gets confused with progression more commonly in high-grade gliomas than low-grade gliomas, and is associated with surgery closer to therapy completion. Careful histological assessment thus may help guide management.

Identification of ATF3 as a novel protective signature of quiescent colorectal tumor cells

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of death in the world. In most cases, drug resistance and tumor recurrence are ultimately inevitable. One obstacle is the presence of chemotherapy-insensitive quiescent cancer cells (QCCs). Identification of unique features of QCCs may facilitate the development of new targeted therapeutic strategies to eliminate tumor cells and thereby delay tumor recurrence. Here, using single-cell RNA sequencing, we classified proliferating and quiescent cancer cell populations in the human colorectal cancer spheroid model and identified ATF3 as a novel signature of QCCs that could support cells living in a metabolically restricted microenvironment. RNA velocity further showed a shift from the QCC group to the PCC group indicating the regenerative capacity of the QCCs. Our further results of epigenetic analysis, STING analysis, and evaluation of TCGA COAD datasets build a conclusion that ATF3 can interact with DDIT4 and TRIB3 at the transcriptional level. In addition, decreasing the expression level of ATF3 could enhance the efficacy of 5-FU on CRC MCTS models. In conclusion, ATF3 was identified as a novel marker of QCCs, and combining conventional drugs targeting PCCs with an option to target QCCs by reducing ATF3 expression levels may be a promising strategy for more efficient removal of tumor cells.

Metastasis Unleashed: Hyposialylation Empowers Chemo-Evasive Circulating Tumor Cell Clusters in Breast Cancer.

Therapy resistance is frequently observed in cancer patients with distant metastases and effective management of metastatic disease remains challenging. Unraveling the cellular mechanisms and molecular targets fueling metastatic spread is crucial for advancing cancer therapies. In a recent issue of Cancer Discovery, Dashzeveg and colleagues revealed that loss of terminal sialylation in glycoproteins within circulating tumor cell clusters is a dynamic process that contributes to cellular dormancy, facilitates evasion of chemotherapy, and enhances metastatic seeding. Furthermore, the study identifies the glycoprotein podocalyxin (PODXL) as a potential target for counteracting the metastasis of quiescent tumor cells associated with paclitaxel treatment in triple-negative breast cancer.

Dynamic Glycoprotein Hyposialylation Promotes Chemotherapy Evasion and Metastatic Seeding of Quiescent Circulating Tumor Cell Clusters in Breast Cancer.

This study discovers that dynamic loss of terminal sialylation in glycoproteins of CTC clusters contributes to the fate of cellular dormancy, advantageous evasion to chemotherapy, and enhanced metastatic seeding. It identifies PODXL as a glycoprotein substrate of ST6GAL1 and a candidate target to counter chemoevasion-associated metastasis of quiescent tumor cells. This article is featured in Selected Articles from This Issue, p. 1949.

Computational Modeling to Determine the Effect of Phenotypic Heterogeneity in Tumors on the Collective Tumor–Immune Interactions

Tumor immunotherapy aims to maintain or enhance the killing capability of CD8+ T cells to clear tumor cells. The tumor-immune interactions affect the function of CD8+ T cells. However, the effect of phenotype heterogeneity of a tumor mass on the collective tumor-immune interactions is insufficiently investigated. We developed the cellular-level computational model based on the principle of cellular Potts model to solve the case mentioned above. We considered how asymmetric division and glucose distribution jointly regulated the transient changes in the proportion of proliferating/quiescent tumor cells in a solid tumor mass. The evolution of a tumor mass in contact with T cells was explored and validated by comparing it with previous studies. Our modeling exhibited that proliferating/quiescent tumor cells, exhibiting distinct anti-apoptotic and suppressive behaviors, redistributed within the domain accompanied by the evolution of a tumor mass. Collectively, a tumor mass prone to a quiescent state weakened the collective suppressive functions of a tumor mass on cytotoxic T cells and triggered a decline of apoptosis of tumor cells. Although quiescent tumor cells did not sufficiently do their inhibitory functions, the possibility of long-term survival was improved due to their interior location within a mass. Overall, the proposed model provides a useful framework to investigate collective-targeted strategies for improving the efficiency of immunotherapy.

Abstract 5947: Tumor cells escape cell death after radiotherapy by senescence - a mechanism that can be counteracted with zinc oxide nanoparticles

Abstract Radiotherapy is still an important pillar for the therapy of head and neck squamous cell carcinoma (HNSCC); however, local recurrences arise in 15-50% of patients. During radiotherapy, there is a risk that individual tumor cells survive and proliferate again after a period of dormancy. Growing evidence suggests that senescence may play a key role in this context. We investigated in vitro radiotherapy-induced senescence and found that quiescent tumor cells survived treatment with 16 Gy. Those cells exhibited characteristics of senescence, yet were able to regain their proliferative capacity, and senescence was associated with radioresistance. During radiotherapy, the cells of the tumor stroma are inevitably irradiated as well. It is already known that cancer-associated fibroblasts (CAFs) can promote tumor progression. However, their contribution to the survival of senescent tumor cells after radiotherapy is unknown so far. Furthermore, the association between radiotherapy-induced senescence and recurrences raises the question of how residual, senescent tumor cells can be eliminated to prevent the development of a recurrence at an early stage. These aspects were the subject of the study presented here. To study the interaction between tumor cells and CAFs during their response to irradiation, both cell types were irradiated with 16 Gy in vitro individually and in co-culture. Subsequently, proliferation, cell death, morphological characteristics, and the secretome were analyzed. Additionally, we investigated whether senescent tumor cells can be targeted by treatment with zinc oxide nanoparticles. We could show that not only the tumor cells but also the CAFs go into a senescent state in response to irradiation, seen by loss of proliferation, increased senescence-associated β-galactosidase activity, and typical morphological characteristics. The secretome analysis revealed that the co-culture of tumor cells with CAFs significantly altered the repertoire of cytokines released upon irradiation, indicating intense communication between the cell types during their response to irradiation. Therefore, it is important for future studies on radiotherapy-induced senescence to include the tumor stroma as well. Finally, we demonstrated that senescent tumor cells were vulnerable to cell death induced by treatment with zinc oxide nanoparticles, which thus could potentially contribute to eliminating residual tumor cells after radiotherapy. This study revealed that radiotherapy-induced senescence in tumor cells and associated fibroblasts could well be a therapeutic problem, as senescent tumor cells are not targetable by radiotherapy anymore. In the future, zinc oxide nanoparticles may help to counteract this escape mechanism to prevent recurrences. Citation Format: Nadine Wiesmann, Rita Gieringer, Johannes Kupka, Jonas Eckrich, Sebahat Kaya, Peer Kaemmerer, Bilal Al-Nawas, Juergen Brieger. Tumor cells escape cell death after radiotherapy by senescence - a mechanism that can be counteracted with zinc oxide nanoparticles [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 5947.

The impact of TP53 status of tumor cells including the type and the concentration of administered 10B delivery agents on compound biological effectiveness in boron neutron capture therapy.

Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or neo vector (SAS/neo) were inoculated subcutaneously into left hind legs of nude mice. After the subcutaneous administration of a 10B-carrier, boronophenylalanine-10B (BPA) or sodium mercaptododecaborate-10B (BSH), at two separate concentrations, the 10B concentrations in tumors were measured using γ-ray spectrometry. The tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) tumor cells, then were administered with BPA or BSH. Subsequently, the tumors were irradiated with reactor neutron beams during the time of which 10B concentrations were kept at levels similar to each other. Following irradiation, cells from some tumors were isolated and incubated with a cytokinesis blocker. The responses of BrdU-unlabeled quiescent (Q) and total (= P + Q) tumor cells were assessed based on the frequencies of micronucleation using immunofluorescence staining for BrdU. In both SAS/neo and SAS/mp53 tumors, the compound biological effectiveness (CBE) values were higher in Q cells and in the use of BPA than total cells and BSH, respectively. The higher the administered concentrations were, the smaller the CBE values became, with a clearer tendency in SAS/neo tumors and the use of BPA than in SAS/mp53 tumors and BSH, respectively. The values for BPA that delivers into solid tumors more dependently on uptake capacity of tumor cells than BSH became more alterable. Tumor micro-environmental heterogeneity might partially influence on the CBE value. The CBE value can be regarded as one of the indices showing the level of intratumor heterogeneity.

STEM-27. MECHANOSENSITIVE BRAIN TUMOR CELLS CONSTRUCT BLOOD-TUMOR BARRIER TO MASK CHEMOSENSITIVITY

Abstract Two major obstacles in brain cancer treatment are the blood-tumor barrier (BTB) and quiescent tumor cells. Here we show that Sox2+ tumor cells directly project cellular processes to ensheathe capillaries in medulloblastoma (MB), a process that depends on the mechanosensitive ion channel Piezo2. MB develops a tissue stiffness gradient as a function of distance to capillaries. Sox2+ tumor cells perceive substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion to promote cellular process growth and cell surface sequestration of β-Catenin. Piezo2 knockout reverses WNT/β-Catenin signaling states between Sox2+ tumor cells and endothelial cells, compromises the BTB, reduces the quiescence of Sox2+ tumor cells, and markedly enhances MB response to chemotherapy. Our study reveals that mechanosensitive tumor cells construct the BTB to mask tumor chemosensitivity. Targeting Piezo2 addresses the BTB and tumor quiescence properties that underlie therapy failures in brain cancer.

Mechanosensitive brain tumor cells construct blood-tumor barrier to mask chemosensitivity

Major obstacles in brain cancer treatment include the blood-tumor barrier (BTB), which limits the access of most therapeutic agents, and quiescent tumor cells, which resist conventional chemotherapy. Here, we show that Sox2+ tumor cells project cellular processes to ensheathe capillaries in mouse medulloblastoma (MB), a process that depends on the mechanosensitive ion channel Piezo2. MB develops a tissue stiffness gradient as a function of distance to capillaries. Sox2+ tumor cells perceive substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion to promote cellular process growth and cell surface sequestration of β-catenin. Piezo2 knockout reverses WNT/β-catenin signaling states between Sox2+ tumor cells and endothelial cells, compromises the BTB, reduces the quiescence of Sox2+ tumor cells, and markedly enhances the MB response to chemotherapy. Our study reveals that mechanosensitive tumor cells construct the BTB to mask tumor chemosensitivity. Targeting Piezo2 addresses the BTB and tumor quiescence properties that underlie treatment failures in brain cancer.

Abstract 2917: Tumor eradication with T-cells targeted with multivalent chemically self-assembled nanorings

Abstract Due to the heterogeneity of cellular dependences on signaling pathways and the potential development of drug resistance, there is an emerging recognition that future cancer therapy will require individualized drug combinations to produce long term remission. In addition, to truly address metastasis and relapse, methods of targeting quiescent tumor initiating cancer stem cells (CSC), which are resistant to chemotherapy and radiation, is of critical importance. Consequently, new methods that can target both primary tumor cells and CSC are needed. Previously, our group has shown that two dihydrofolate reductase molecules (DHFR2) fused to distinct targeting ligands can spontaneously self-assemble upon the addition of a chemical dimerizer (bis-methotrexate (BisMTX)) into highly stable, multivalent, chemically self-assembled nanorings (CSANs). When an anti-CD3 scFv is fused, the bispecific CSANs rapidly (min) and stably (days) bind to CD3 on T-cell membranes. Consequently, we refer to the modified T-cells as Prosthetic Antigen Receptor (PAR) T-cells. A unique feature of our approach is the ability to remove the CSANs from the T-cells by incubation with the FDA-approved antibiotic trimethoprim, allowing us to deactivate the modified cells pharmacologically. In addition, we have shown that the CSANs have negligible immunogenicity in mice and do not induce naïve human T-cell anergy (i.e, unresponsiveness).Recently, we have prepared anti-EpCAM/anti-CD3 CSANs, which target triple negative cancer (TNBC) primary tumor cells, and anti-CD133/anti-CD3-CSANs, which target TNBC cancer stem cells. The bispecific CSANs rapidly (min) and stably (days) bind to CD3 on T-cell membranes, thus either anti-EpCAM or anti-CD133 PAR T-cells. Upon incubation of CD133+ or EpCAM+ TNBC cells with the CSANs rapid and selective cell killing was observed. We have also demonstrated with an orthotopic murine cancer model that anti-EpCAM CSANs significantly reduce tumor size, while anti-CD133 CSANs block tumor proliferation. When dosed with a combination of anti-EpCAM CSANs and anti-CD133 CSANs tumor T-cell induced eradication of the tumors is observed. Further analysis revealed that initial tumor growth kinetics is dependent on the percentage of CD133+ tumor cells and that regardless of the starting point, the number of CD133+ tumor cells reaches a homeostatic percentage in the tumor. Whether CD133+ tumor cells arise from self-renewal or de-differentiation is yet to be determined. Regardless, dosing with either CSANs was found to be non-toxic. Taken together, our results demonstrate that CSAN modified T-cells targeting both primary tumor and CSC are necessary for TNBC tumor eradication and that CD133+ TNBC tumor cells maybe be necessary and sufficient for TNBC tumor initiation and proliferation. Citation Format: Carston R. Wagner. Tumor eradication with T-cells targeted with multivalent chemically self-assembled nanorings [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 2917.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.