Primary Tumor Cells Research Articles

Olanzapine suppresses mPFC activity-norepinephrine releasing to alleviate CLOCK-enhanced cancer stemness under chronic stress

BackgroundOlanzapine (OLZ) reverses chronic stress-induced anxiety. Chronic stress promotes cancer development via abnormal neuro-endocrine activation. However, how intervention of brain-body interaction reverses chronic stress-induced tumorigenesis remains elusive.MethodsKrasLSL−G12D/WT lung cancer model and LLC1 syngeneic tumor model were used to study the effect of OLZ on cancer stemness and anxiety-like behaviors. Cancer stemness was evaluated by qPCR, western-blotting, immunohistology staining and flow-cytometry analysis of stemness markers, and cancer stem-like function was assessed by serial dilution tumorigenesis in mice and extreme limiting dilution analysis in primary tumor cells. Anxiety-like behaviors in mice were detected by elevated plus maze and open field test. Depression-like behaviors in mice were detected by tail suspension test. Anxiety and depression states in human were assessed by Hospital Anxiety and Depression Scale (HADS). Chemo-sensitivity of lung cancer was assessed by in vivo syngeneic tumor model and in vitro CCK-8 assay in lung cancer cell lines.ResultsIn this study, we found that OLZ reversed chronic stress-enhanced lung tumorigenesis in both KrasLSL−G12D/WT lung cancer model and LLC1 syngeneic tumor model. OLZ relieved anxiety and depression-like behaviors by suppressing neuro-activity in the mPFC and reducing norepinephrine (NE) releasing under chronic stress. NE activated ADRB2-cAMP-PKA-CREB pathway to promote CLOCK transcription, leading to cancer stem-like traits. As such, CLOCK-deficiency or OLZ reverses NE/chronic stress-induced gemcitabine (GEM) resistance in lung cancer. Of note, tumoral CLOCK expression is positively associated with stress status, serum NE level and poor prognosis in lung cancer patients.ConclusionWe identify a new mechanism by which OLZ ameliorates chronic stress-enhanced tumorigenesis and chemoresistance. OLZ suppresses mPFC-NE-CLOCK axis to reverse chronic stress-induced anxiety-like behaviors and lung cancer stemness. Decreased NE-releasing prevents activation of ADRB2-cAMP-PKA-CREB pathway to inhibit CLOCK transcription, thus reversing lung cancer stem-like traits and chemoresistance under chronic stress.

G-quadruplexes are a source of vulnerability in BRCA2 deficient granule cell progenitors and medulloblastoma.

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2 Δex3-4 ;Trp53 -/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

Promising approach for targeting ROBO1 with CAR NK cells to combat ovarian cancer primary tumor cells and organoids.

Aim: This study aimed to explore using peripheral blood mononuclear cell (PBMC)-derived chimeric antigen receptor (CAR) NK cells targeting ROBO1 as a personalized medicine approach for ovarian cancer. Methods: A two-step strategy generated ROBO1-targeted CAR NK cells from PBMCs of ovarian cancer patients. Efficacy was evaluated using xCELLigence RTCA, CCK-8 and Live/Dead fluorescence assays. Results: ROBO1-NK cells exhibited higher efficiency in eradicating primary ovarian cancer cells and lysing ovarian tumor organoids compared with primary NK cells without ROBO1-CAR modification. Conclusion: These findings highlight the potential of developing ROBO1-targeted CAR-NK cells from patients' PBMCs as a personalized treatment option for ovarian cancer.

An easy-to-perform protocol for culturing primary murine lung tumor cells as organoids

Cancer research involves significant animal consumption and suffering. Tumor cells can be differentiated in vitro into three-dimensional organoids that resemble the primary tumor. In basic cancer research, however, tumor organoids are usually only used alongside animal experiments. We have established an easy-to-perform protocol that allows to culture KRAS-driven lung tumor cells as organoids for extended periods of time. Like the corresponding tumors in mice, the organoids produce surfactant protein C but no markers of airway epithelial cells (e.g. SCGB1A1, KRT5). The organoids can be passaged as single cell suspensions. Our organoid model contributes to replace animal experiments with cell culture systems and can be used for drug testing or functional studies in cancer research.

Tumor-targeting and activatable biomimetic nanococktails synergistically regulate immune responses for spatiotemporal immunotherapy of low immunogenic solid tumors

Cancer immunotherapy is generally limited by the low immunogenicity of tumor microenvironments (TME). Thus, regulating immune responses in low immunogenic solid tumors is critical for improving immunotherapeutic outcomes. Here, the tumor-targeting and activatable biomimetic nanococktails have been engineered to synergistically and systemically activate a cascade of immune responses and downregulate immunosuppressive T cells for spatiotemporal immunotherapy of low immunogenic solid tumors. The nanococktails had core-shell structures, where the photosensitizers, STING agonists and indoleamine 2,3-dioxygen-ase 1 (IDO1) inhibitors were encapsulated within the core, which can disassociate when responding to the low pH in tumors to disrupt the particle shells and cellular membranes for precision drug delivery and release. The ligands anchored on the shell of nanococktails could efficiently target the receptors that were identified to be highly expressed in the breast cancer cells, facilitating efficient intracellular drug delivery and synergistically inducing anticancer immunity and mitochondrial damage. By intravenous (i.v.) injection, the nanococktails demonstrated high accumulation in triple-negative breast cancer (TNBC), endowing precision diagnosis and effective elimination of primary TNBC tumors. The synergistic effects of STING activation and metalloimmunotherapy induced by nanococktails can amplify systemic immune responses and downregulate immunosuppressive regulatory T cells (Tregs) in primary tumors, lymph nodes and distant tumors, which spatiotemporally inhibited distant tumors and lung metastasis of TNBC with promoted survival rates. This study presents a promising strategy for leveraging nanococktails to regulate immune responses for effective immunotherapy.

A machine learning and drug repurposing approach to target ferroptosis in colorectal cancer stratified by sex and KRAS.

The landscape of sex differences in Colorectal Cancer (CRC) has not been well characterized with respect to the mechanisms of action for oncogenes such as KRAS. However, our recent study showed that tumors from male patients with KRAS mutations have decreased iron-dependent cell death called ferroptosis. Building on these findings, we further examined ferroptosis in CRC, considering both sex of the patient and KRAS mutations, using public databases and our in-house CRC tumor cohort. Through subsampling inference and variable importance analysis (VIMP), we identified significant differences in gene expression between KRAS mutant and wild type tumors from male patients. These genes suppress (e.g., SLC7A11 ) or drive (e.g., SLC1A5 ) ferroptosis, and these findings were further validated with Gaussian mixed models. Furthermore, we explored the prognostic value of ferroptosis regulating genes and discovered sex- and KRAS-specific differences at both the transcriptional and metabolic levels by random survival forest with backward elimination algorithm (RSF-BE). Of note, genes and metabolites involved in arginine synthesis and glutathione metabolism were uniquely associated with prognosis in tumors from males with KRAS mutations. Additionally, drug repurposing is becoming popular due to the high costs, attrition rates, and slow pace of new drug development, offering a way to treat common and rare diseases more efficiently. Furthermore, increasing evidence has shown that ferroptosis inhibition or induction can improve drug sensitivity or overcome chemotherapy drug resistance. Therefore, we investigated the correlation between gene expression, metabolite levels, and drug sensitivity across all CRC primary tumor cell lines using data from the Genomics of Drug Sensitivity in Cancer (GDSC) resource. We observed that ferroptosis suppressor genes such as DHODH , GCH1 , and AIFM2 in KRAS mutant CRC cell lines were resistant to cisplatin and paclitaxel, underscoring why these drugs are not effective for these patients. The comprehensive map generated here provides valuable biological insights for future investigations, and the findings are supported by rigorous analysis of large-scale publicly available data and our in-house cohort. The study also emphasizes the potential application of VIMP, Gaussian mixed models, and RSF-BE models in the multi-omics research community. In conclusion, this comprehensive approach opens doors for leveraging precision molecular feature analysis and drug repurposing possibilities in KRAS mutant CRC.

Mechanisms underlying neutrophils adhesion to triple-negative breast cancer cells via CD11b-ICAM1 in promoting breast cancer progression

BackgroundTriple-negative breast cancer (TNBC) is recognized as the most aggressive and immunologically infiltrated subtype of breast cancer. A high circulating neutrophil-to-lymphocyte ratio (NLR) is strongly linked to a poor prognosis among patients with breast cancer, emphasizing the critical role of neutrophils. Although the involvement of neutrophils in tumor metastasis is well documented, their interactions with primary tumors and tumor cells are not yet fully understood.MethodsClinical data were analyzed to investigate the role of neutrophils in breast cancer. In vivo mouse model and in vitro co-culture system were used for mechanism researches. Blocking experiments were further performed to identify therapeutic agents against TNBC.ResultsTNBC cells secreted GM-CSF to sustain the survival of mature neutrophils and upregulated CD11b expression. Through CD11b, neutrophils specifically binded to ICAM1 on TNBC cells, facilitating adhesion. Transcriptomic sequencing combined with human and murine functional experiments revealed that neutrophils, through direct CD11b-ICAM1 interactions, activated the MAPK signaling pathway in TNBC cells, thereby enhancing tumor cell invasion and migration. Atorvastatin effectively inhibited ICAM1 expression in tumor cells, and tumor cells with ICAM1 knockout or treated with atorvastatin were unresponsive to neutrophil activation. The MAPK pathway and MMP9 expression were significantly inhibited in the tumor tissues of TNBC patients treated with atorvastatin.ConclusionsTargeting CD11b-ICAM1 with atorvastatin represented a potential clinical approach to reduce the malignant characteristics of TNBC.Graphical

STEM-03. A NOVEL MOUSE MODEL OF CHOROID PLEXUS CARCINOMA UNRAVELS SEQUENTIAL DEDIFFERENTIATION OF HINDBRAIN CHOROID PLEXUS TISSUE AND REVEALS NEW THERAPEUTIC PROSPECTS

Abstract BACKGROUND Choroid Plexus tumors constitute 10-20% of brain tumors in the first year of life. Among those, Choroid Plexus Carcinomas (CPC) pose a significant challenge due to their aggressiveness, resulting in poor survival rates and long-term adverse effects stemming from intense therapeutic approaches. Although loss of p53 and Sonic hedgehog signaling have been implicated in CPC development, a deeper biological understanding is needed to expand mechanistic insights and advance targeted therapies. METHODS We generated a novel mouse model with a simultaneous loss of TP53 and overexpression of MYCN and Gli2 in central nervous system precursors (hGFAP-cre::P53Fl/Fl::lsl-MYCN::lsl-Gli2). Resulting tumors were histologically characterized and compared to human CPC. Moreover, spatial transcriptomic data were generated for molecular characterization. Finally, mouse tumor-derived cell lines were employed for differentiation analysis and therapeutic testing. RESULTS Examinations at different developmental stages revealed brain tumors in the choroid plexus as well as smaller lesions in the subcallosal cortex with a penetrance of 80% within 18 days. We observed a gradual dedifferentiation of hindbrain CP tissue from day 5 onwards, indicating a continuous developmental sequence corresponding with expanding tumor growth. The mouse model recapitulates early tumor development, aggressive and metastatic growth along with histological features of human tumors. Spatial transcriptomic analysis highlighted chromosomal aberrations as well as other features known from human CPCs and emphasized the disturbance of ciliary pathways in tumor development. In vitro studies revealed rapidly growing tumor cells with stem cell-like differentiation potential and high sensitivity to targeted therapies. CONCLUSION Through in-depth analysis, including multiple data types, we show that our novel CPC mouse model closely resembles human tumors. Our tumors reveal a strict pattern of dedifferentiation implying similar mechanisms in humans. Additionally, primary mouse tumor cells in culture facilitate further in vitro analysis, and newly generated spatial transcriptomic data enables further insights into tumor biology.

Magnetic Particle Imaging Reveals that Iron-Labeled Extracellular Vesicles Accumulate in Brains of Mice with Metastases.

The incidence of breast cancer remains high worldwide and is associated with a significant risk of metastasis to the brain that can be fatal; this is due, in part, to the inability of therapeutics to cross the blood-brain barrier (BBB). Extracellular vesicles (EVs) have been found to cross the BBB and further have been used to deliver drugs to tumors. EVs from different cell types appear to have different patterns of accumulation and retention as well as the efficiency of bioactive cargo delivery to recipient cells in the body. Engineering EVs as delivery tools to treat brain metastases, therefore, will require an understanding of the timing of EV accumulation and their localization relative to metastatic sites. Magnetic particle imaging (MPI) is a sensitive and quantitative imaging method that directly detects superparamagnetic iron. Here, we demonstrate MPI as a novel tool to characterize EV biodistribution in metastatic disease after labeling EVs with superparamagnetic iron oxide (SPIO) nanoparticles. Iron-labeled EVs (FeEVs) were collected from iron-labeled parental primary 4T1 tumor cells and brain-seeking 4T1BR5 cells, followed by injection into the mice with orthotopic tumors or brain metastases. MPI quantification revealed that FeEVs were retained for longer in orthotopic mammary carcinomas compared to SPIOs. MPI signal due to iron could only be detected in brains of mice bearing brain metastases after injection of FeEVs, but not SPIOs, or FeEVs when mice did not have brain metastases. These findings indicate the potential use of EVs as a therapeutic delivery tool in primary and metastatic tumors.

Developmental and therapeutic implications of IL4ra expression for rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a solid tumor whose metastatic progression can be accelerated through interleukin-4 receptor alpha (Il4ra) mediated interaction with normal muscle stem cells (satellite cells). To understand the function of Il4ra in this tumor initiation phase of RMS, we conditionally deleted Il4ra in genetically-engineered RMS mouse models. Nullizygosity of Il4ra altered the latency, site and/or stage distribution of RMS tumors compared to IL4RA intact models. Primary tumor cell cultures taken from the genetically-engineered models then used in orthotopic allografts further defined the interaction of satellite cells and RMS tumor cells in the context of tumor initiation: in alveolar rhabdomyosarcoma (ARMS), satellite cell co-injection was necessary for Il4ra null tumor cells engraftment, whereas in embryonal rhabdomyosarcoma (ERMS), satellite cell co-injection decreased latency of engraftment of Il4ra wildtype tumor cells but not Il4ra null tumor cells. When refocusing on Il4ra wildtype tumors by single cell sequencing and cytokine studies, we have uncovered a putative signaling interplay of Il4 from T-lymphocytes being received by Il4ra + rhabdomyosarcoma tumor cells, which in turn express Ccl2, the ligand for Ccr2 and Ccr5. Taken together, these results suggest that mutations imposed during tumor initiation have different effects than genetic or therapeutic intervention imposed once tumors are already formed. We also propose that CCL2 and its cognate receptors CCR2 and/or CCR5 are potential therapeutic targets in Il4ra mediated RMS progression.

Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection

Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model.

A phase 1, multi-center, safety, feasibility, and preliminary efficacy study evaluating a single dose of UNO101 in relapsed or refractory, unresectable, primary, or metastatic cutaneous and subcutaneous malignancies.

TPS2686 Background: Tumor ablation is a minimally invasive technique commonly used to treat solid tumors in the liver, kidney, bone, and lung and is usually based on thermal and nonthermal approaches. Local and in-situ tumor ablation methods demonstrate enhanced anti-tumor immune responses resulting in the destruction of residual malignant cells in primary tumors and distant metastases. Nitric oxide (NO) is a colorless gas and a short-lived free radical. It is a ubiquitous, endogenously generated gas implicated in the homeostatic regulation of physiological processes. Preclinical studies evaluating the effect of high concentration exogenously administered NO demonstrated its anti-cancer properties and suggested that it may serve as a potent tumoricidal agent. We have previously shown that treating mouse colon carcinoma (CT26) tumor-bearing mice with ultra-high concentrations of nitric oxide (UNO101) upregulates innate and adaptive immune cells both locally and systemically. Beyond Cancer is currently conducting a first-in-human, first-in-class, Phase 1 safety, feasibility, and preliminary efficacy clinical study of UNO101 at multiple institutions in Israel. Methods: The study is a 2-part Phase 1 trial with a Dose Escalation and a Dose Expansion portion (NCT05351502). A conventional 3+3 dose escalation will evaluate three cohorts of UNO101 single dose: 25,000, 50,000 and 100,000 parts per million (PPM) delivered intratumorally over 5 minutes in subjects with an ECOG PS of 0–3, at least 3 months of life expectancy, with relapsed or refractory unresectable primary or metastatic cutaneous and subcutaneous measurable solid tumors being eligible for enrollment. Upon determination of the maximum tolerated dose or biological effective dose, whichever occurs first, the proposed recommended Phase 2 dose will be further evaluated in the Dose Expansion portion of the study. RECIST version 1.1 and iRECIST will be utilized to assess the rate of malignant tumor response after UNO administration and toxicity will be graded per NCI CTCAE version 5.0. Evaluation of response per itRECIST will also be explored. Up to thirty-eight enrolled subjects are anticipated. Cohort 1, 25,000 PPM have been completed without a reported DLT. Enrollment to Cohort 2, 50,000 PPM, began in January 2024. This study was approved by Israel Ministry of Health (IMOH) as well as the participating institution’s Ethics Board. Written informed consent was obtained for all enrolled subjects and a copy of the written consent is available for review. Clinical trial information: NCT05351502 .

Transcriptional mechanisms of phenotypic plasticity in glioblastoma.

e14027 Background: Primary glioblastoma (GBM) tumor cells exhibit plasticity and can adopt different cellular states that exhibit dynamic transitions between states. This could be one of the mechanisms for treatment escape and leads to poor prognosis in GBM. In this regard, we sought to uncover transcriptional regulatory mechanisms responsible for this phenotypic plasticity in GBM. Methods: Standard of care (SOC) treatment (radiation and temozolomide) modulates transcriptional regulatory networks (TRNs) of the tumor cells and drives treatment resistance. Since TRNs could be key mechanistic drivers for state transitions, integrated single-cell RNA sequencing (scRNAseq) data of primary and relapse patient samples (11) were analyzed with scMINER. Our method uses a systems biology framework to identify cell states (St_1 to St_n) in terms of regulatory networks and their activities. Clustering of co-regulated gene modules (regulons), enabled the identification of transcriptional programs and regulatory mechanisms that specify tumor cell states. Results: Activity patterns of 38 transcriptional programs clustered tumor cells from the patients into 21 states. Preliminarily, we observed common regulatory network cell states (St_1, St_2, St_5, St_6) across patient tumors. Additionally, primary tumor cells of a patient (UW14) transitioned from initial cell state(s) observed across multiple patients (e.g., St_1) to a recurrent state (e.g., St_9) through a trajectory driven by transcription factors (TFs). Herein, scMINER regulatory network inference method also defines master TFs (TFs that regulate the majority of the other TFs in the network) that were used to characterize initial (St_1) or recurrent (St_9) cell states. Moreover, certain transcription factors (22) and their co-expressed target genes (regulons) also exhibit variation in expression across the pseudotime trajectory (from St_1 to St_9) including masterTFs such as SOX9 and MECOM. Conclusions: Therefore, we observed that shared cell states may exist across patients (primary and relapse) although in different proportions. Our analysis suggests a de-differentiation trajectory from neuronal cells to developing GBM stem cells and predicts transcription factors that could be playing an important role therein. Moreover, similar transcriptional regulatory mechanisms may promote transitions between primary and recurrent tumor cell states that exhibit phenotypic plasticity.

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

The temporal progression of lung immune remodeling during breast cancer metastasis

Tumor metastasis requires systemic remodeling of distant organ microenvironments that impacts immune cell phenotypes, population structure, and intercellular communication. However, our understanding of immune phenotypic dynamics in the metastatic niche remains incomplete. Here, we longitudinally assayed lung immune transcriptional profiles in the polyomavirus middle T antigen (PyMT) and 4T1 metastatic breast cancer models from primary tumorigenesis, through pre-metastatic niche formation, to the final stages of metastatic outgrowth at single-cell resolution. Computational analyses of these data revealed a TLR-NFκB inflammatory program enacted by both peripherally derived and tissue-resident myeloid cells that correlated with pre-metastatic niche formation and mirrored CD14+ "activated" myeloid cells in the primary tumor. Moreover, we observed that primary tumor and metastatic niche natural killer (NK) cells are differentially regulated in mice and human patient samples, with the metastatic niche featuring elevated cytotoxic NK cell proportions. Finally, we identified cell-type-specific dynamic regulation of IGF1 and CCL6 signaling during metastatic progression that represents anti-metastatic immunotherapy candidate pathways.

3D tumor spheroids promote activation, expansion, and anti-tumor effects of tumor-infiltrating lymphocytes in vitro

The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro, aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3+/CD8+ cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3+/CD8+ cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3+/CD8+ cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.

Drug screening on digital microfluidics for cancer precision medicine

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells

Background: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. Methods: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. Results: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. Conclusion: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

Methods for assessing and removing non-specific photoimmunotherapy damage in patient-derived tumor cell culture models.

Tumor-targeted, activatable photoimmunotherapy (taPIT) has been shown to selectively destroy tumor in a metastatic mouse model. However, the photoimmunoconjugate (PIC) used for taPIT includes a small fraction of non-covalently associated (free) benzoporphyrin derivative (BPD), which leads to non-specific killing invitro. Here, we report a new treatment protocol for patient-derived primary tumor cell cultures ultrasensitive to BPD photodynamic therapy (BPD-PDT). Based on free BPD efflux dynamics, the updated invitro taPIT protocol precludes non-specific BPD-PDT by silencing the effect of free BPD. Following incubation with PIC, incubating cells with PIC-free medium allows time for expulsion of free BPD whereas BPD covalently bound to PIC fragments is retained. Administration of the light dose after the intracellular free BPD drops below the threshold for inducing cell death helps to mitigate non-specific damage. In this study, we tested two primary ovarian tumor cell lines that are intrinsically chemoresistant, yet ultrasensitive to BPD-PDT such that small amounts of free BPD (afew percent of the total BPD dose) lead to potent induction of cell death upon irradiation. The modifications in the protocol suggested here improve invitro taPIT experiments that lack invivo mechanisms of free BPD clearance (i.e., lymph and blood flow).

Extremely low-frequency electromagnetic field (ELF-EMF) induced alterations in gene expression and cytokine secretion in clear cell renal carcinoma cells.

The study aimed to investigate the influence of extremely low-frequency electromagnetic fields (ELF-EMF) on clear cell renal cell carcinoma (ccRCC) by assessing alterations in gene expression and the secretion of cytokines and chemokines. Three ccRCC cell lines (786-O, 769-P, and CAKI-1) and a healthy HEK293 cell line were subjected to ELF-EMF exposure (frequency 50 Hz, magnetic field strength 4.5 mT) for 30 min daily for 5 days. The study examined the expression of ADAM28, NCAM1, and VEGFC genes, along with the secretion of 30 cytokines and chemokines. Notably, primary tumor-derived cell lines, but not those from metastatic sites, exhibited ADAM28 gene expression, which increased following ELF-EMF exposure. A statistically significant reduction in VEGFC gene expression was observed in 769-P cells after ELF-EMF exposure. Additionally, NCAM1 gene expression was upregulated in HEK293, 769-P, and 786-O cells, representing normal embryonic kidney cells and primary tumor cells, but not in CAKI-1 cells, which model metastatic sites. After EMF exposure, there was a statistically significant decrease in transforming growth factor β1 (TGF-β1) concentration in the cell culture supernatants of HEK293 and CAKI-1 cell lines, with no other significant changes in the secretion of tested cytokines. Given the study's findings and available research, caution is warranted when drawing conclusions about the potential inhibitory effect of ELF-EMF on ccRCC progression. Standardization of experimental models is imperative when assessing the effects of EMF in a human context. Med Pr Work Health Saf. 2024;75(2):133-141.