Tumor Chemoresistance Research Articles

Cancer-associated fibroblasts: protagonists of the tumor microenvironment in gastric cancer.

Enhanced knowledge of the interaction of cancer cells with their environment elucidated the critical role of tumor microenvironment in tumor progression and chemoresistance. Cancer-associated fibroblasts act as the protagonists of the tumor microenvironment, fostering the metastasis, stemness, and chemoresistance of cancer cells and attenuating the anti-cancer immune responses. Gastric cancer is one of the most aggressive cancers in the clinic, refractory to anti-cancer therapies. Growing evidence indicates that cancer-associated fibroblasts are the most prominent risk factors for a poor tumor immune microenvironment and dismal prognosis in gastric cancer. Therefore, targeting cancer-associated fibroblasts may be central to surpassing resistance to conventional chemotherapeutics, molecular-targeted agents, and immunotherapies, improving survival in gastric cancer. However, the heterogeneity in cancer-associated fibroblasts may complicate the development of cancer-associated fibroblast targeting approaches. Although single-cell sequencing studies started dissecting the heterogeneity of cancer-associated fibroblasts, the research community should still answer these questions: "What makes a cancer-associated fibroblast protumorigenic?"; "How do the intracellular signaling and the secretome of different cancer-associated fibroblast subpopulations differ from each other?"; and "Which cancer-associated fibroblast subtypes predominate specific cancer types?". Unveiling these questions can pave the way for discovering efficient cancer-associated fibroblast targeting strategies. Here, we review current knowledge and perspectives on these questions, focusing on how CAFs induce aggressiveness and therapy resistance in gastric cancer. We also review potential therapeutic approaches to prevent the development and activation of cancer-associated fibroblasts via inhibition of CAF inducers and CAF markers in cancer.

Basal–epithelial subpopulations underlie and predict chemotherapy resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, characterized by extensive intratumoral heterogeneity, high metastasis, and chemoresistance, leading to poor clinical outcomes. Despite progress, the mechanistic basis of these aggressive behaviors remains poorly understood. Using single-cell and spatial transcriptome analysis, here we discovered basal epithelial subpopulations located within the stroma that exhibit chemoresistance characteristics. The subpopulations are defined by distinct signature genes that show a frequent gain in copy number and exhibit an activated epithelial-to-mesenchymal transition program. A subset of these genes can accurately predict chemotherapy response and are associated with poor prognosis. Interestingly, among these genes, elevated ITGB1 participates in enhancing intercellular signaling while ACTN1 confers a survival advantage to foster chemoresistance. Furthermore, by subjecting the transcriptional signatures to drug repurposing analysis, we find that chemoresistant tumors may benefit from distinct inhibitors in treatment-naive versus post-NAC patients. These findings shed light on the mechanistic basis of chemoresistance while providing the best-in-class biomarker to predict chemotherapy response and alternate therapeutic avenues for improved management of TNBC patients resistant to chemotherapy.

Hypoxia-induced TREM1 promotes mesenchymal-like states of glioma stem cells via alternatively activating tumor-associated macrophages

The mesenchymal subtype of glioblastoma (GBM) cells characterized by aggressive invasion and therapeutic resistance is thought to be dependent on cell-intrinsic alteration and extrinsic cellular crosstalk. Tumor-associated macrophages (TAMs) are pivotal in tumor progression, chemo-resistance, angiogenesis, and stemness maintenance. However, the impact of TAMs on the shifts in glioma stem cells (GSCs) states remains largely uncovered. Herein, we showed that the triggering receptor expressed on myeloid cells-1 (TREM1) preferentially expressed by M2-like TAMs and induced GSCs into mesenchymal-like states by modulating the secretion of TGFβ2, which activated the TGFβR/SMAD2/3 signaling in GSCs. Furthermore, we demonstrated that TREM1 was transcriptionally regulated by HIF1a under the hypoxic environment and thus promoted an immunosuppressive type of TAMs via activating the TLR2/AKT/mTOR/c-MYC axis. Collectively, this study reveals that cellular communication between TAMs and GSCs through the TREM1-mediated TGFβ2/TGFβR axis is involved in the mesenchymal-like transitions of GSCs. Our study provides valuable insights into the regulatory mechanisms between the tumor immune microenvironment and the malignant characteristics of GBM, which can lead to potential novel strategies targeting TAMs for tumor control.

Reversal of Chemoresistance via Staged Liberation of Chemodrug and siRNA in Hierarchical Response to ROS Gradient.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) often leads to the failure of antitumor chemotherapy, and codelivery of chemodrug with P-gp siRNA (siP-gp) represents a promising approach for treating chemoresistant tumors. To maximize the antitumor efficacy, it is desired that the chemodrug be latently released upon completion of siP-gp-mediated gene silencing, which however, largely remains an unmet demand. Herein, core-shell nanocomplexes (NCs) are developed to overcome MDR via staged liberation of siP-gp and chemodrug (doxorubicin, Dox) in hierarchical response to reactive oxygen species (ROS) concentration gradients. The NCs are constructed from mesoporous silica nanoparticles (MSNs) surface-decorated with cRGD-modified, PEGylated, ditellurium-crosslinked polyethylenimine (RPPT), wherein thioketal-linked dimeric doxorubicin (TK-Dox2) and photosensitizer are coencapsulated inside MSNs while siP-gp is embedded in the RPPT polymeric layer. RPPT with ultrahigh ROS-sensitivity can be efficiently degraded by the low-concentration ROS inside cancer cells to trigger siP-gp release. Upon siP-gp-mediated gene silencing and MDR reversal, light irradiation is performed to generate high-concentration, lethal amount of ROS, which cleaves thioketal with low ROS-sensitivity to liberate the monomeric Dox. Such a latent release profile greatly enhances Dox accumulation in Dox-resistant cancercells (MCF-7/ADR) in vitro and in vivo, which cooperates with the generated ROS to efficiently eradicate MCF-7/ADR xenograft tumors.

Abstract B086: Novel myeloid activation therapy promotes regression of metastatic ovarian cancer

Abstract Significance: Most ovarian cancer (OC) patients ultimately succumb to drug-resistant intraperitoneal metastases. There is an unmet need to develop novel therapies against metastatic OC. Whether activating myeloid cells can promote OC regression has not been explored extensively. The purpose of this study is to develop a novel immunotherapy that activates myeloid cells with the goal of eradicating metastatic OC. Methods: In this study, we mainly relied on syngeneic mouse models of metastatic OC. To model metastatic OC in mice, we performed intraperitoneal injection of clinically relevant murine OC cell lines that were recently established by Dr. Robert Weinberg’s group, including homologous recombination (HR)-proficient KPCA cells (KRASG12VTrp53R172HCcne1OEAkt2OE ) and HR-deficient BPPNM cells (Brca1−/−Trp53R172HPten−/− Nf1−/−MycOE ). To activate myeloid cells, we used b-glucan and interferon (IFN) γ. b-glucan is a yeast cell wall polysaccharide that is currently in clinical trials to treat multiple cancers (NCT05159778). It canonically activates myeloid cells through the Dectin-1-spleen tyrosine kinase (SYK) pathway and induces infiltration of monocytes and neutrophils. IFNγ activates macrophages to an anti-tumor status. We investigated whether and how b-glucan affects metastatic OC with or without IFNγ using bioluminescence imaging, confocal imaging, flow cytometry, and single-cell RNA sequencing (scRNA seq). Finally, we tested whether combining myeloid cell activation with carboplatin could eradicate metastatic OC.Results: First, we found that b-glucan alone was highly efficient in clearing ascites. b-glucan captured free-floating OC cells into solid nodules within 5 hours after its injection through two non-redundant and equally important pathways: (1) an unexpected Dectin-1-SYK-independent, heparin-sensitive pathway that was mediated by resident macrophages in the peritoneal fluid; (2) a Dectin-1-SYK-PAD4-dependent pathway that was mediated by neutrophil extracellular traps in the omentum. Second, we found that combining b-glucan with IFNγ (b-glucan/IFNγ) not only cleared ascites but also reduced total metastases compared to PBS-, b-glucan-, or IFNγ-treated mice. This anti-tumor immunity required T cells and non-tumor IFNγ signaling in the host. Third, using scRNA seq, we found that b-glucan/IFNγ induced the enrichment of unique subsets of tumoricidal neutrophils and macrophages compared to other groups. Interestingly, the novel macrophage subset selectively expressed an interleukin (IL)-12 family cytokine, IL-27. We found that blocking IL-27 significantly impaired the anti-tumor response. Finally, combining b-glucan/IFNγ with carboplatin nearly eradicated chemoresistant KPCA tumors and chemosensitive BPPNM tumors. Patients with high expression of the genes identified in the novel myeloid cell subsets correlate with better overall survival. Conclusion: We have identified a novel combination therapy of myeloid activation and chemotherapy that could potentially transform treatments against metastatic OC. Citation Format: Brennah Murphy, Taito Miyamoto, Bryan Manning, Toshitha Kannan, Anastasia Ghilkovsky, Yulia Nefedova, Daniel Claiborne, Rugang Zhang, Andrew Kossenkov, Nan Zhang. Novel myeloid activation therapy promotes regression of metastatic ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B086.

Abstract A003: Characterizing DNA methylation patterns driving chemotherapy resistance in ovarian high-grade serous carcinoma

Abstract To characterize the role of DNA methylation (DNAm) in chemotherapy response, we analyzed whole-genome DNAm data integrated with transcriptome from 67 samples of 17 patients with ovarian high grade serous carcinoma (HGSC) belonging to the prospective, longitudinal, multi-region DECIDER trial (NCT04846933). We used a novel decomposition method, which allows separating the effects of tumor microenvironment and treatment, tissue, and patient specific changes, facilitating the integration of the heterogeneous collection of samples. We firstly characterized DNAm alterations between tissue sites. Our results show that the cancer spread from tubo-ovarian sites to metastatic deposits is accompanied by moderate changes that employ cell junctions and focal adhesions. The most drastic change in the tissue intrinsic DNAm profiles implicated upregulation of members of the ATP-binding-cassette (ABC) family of transporters, including such multidrug resistance-associated genes as ABCC2, ABCC3, and ABCC4 in omentum. We then proceeded to characterize the DNAm changes during treatment. Our results reveal that DNAm plays a distinct role in emergence, driving, and regulation of both preexisting and acquired resistance to chemotherapy. We observed that patient-specific DNAm background shows enormous intra- and intertumor heterogeneity and sustains multiple driver mechanisms to resist chemotherapy-induced cell death already prior to treatment. We also observed that neoadjuvant chemotherapy (NACT) results in emergence of resistance via acquired, tissue specific DNAm alterations in DNA damage repair, apoptosis, cellular senescence, and drug efflux. Pathway analyses show that treatment-naïve tumors from patients with poor treatment outcome have DNAm-induced upregulation of key resistance promoting signaling pathways, such as PI3K/Akt, mTOR, and Rap1 cascades. Hypoxic transcriptional program activated via HIF-1 signaling was significantly altered in poorly responding patients, accompanied by changes in extracellular matrix instigated by integrins ITGB1 and ITGA5. Tumors of patients with refractory disease demonstrated DNAm changes in genes involved in tumor-promoting chronic inflammation driven by elevation of tumor necrosis factor. NACT treatment resulted in very few changes to these DNA aberrations and no acquired mechanisms, suggesting that intrinsic DNAm patient profiles accommodate multiple drivers for tumor progression and chemoresistance. In conclusion, using a large whole-sequencing DNAm dataset we elucidated the major underlying mechanisms of acquired platinum resistance, which provide possible targets for improving platinum sensitivity. Validation of these results and the intrinsic ability of HGSC to resist platinum-based treatment is currently on-going in our recently sequenced DNAm batch consisting of 400 samples of 135 HGSC patients. Citation Format: Alexandra Lahtinen, Giovanni Marchi, Kari Lavikka, Giulia Micoli, Susanna Holmström, Elsi Pöllänen, Ilari Maarala, Daria Afenteva, Yilin Li, Taru A. Muranen, Jaana Oikkonen, Kaisa Huhtinen, Anni Virtanen, Johanna Hynninen, Antti Häkkinen, Sampsa Hautaniemi. Characterizing DNA methylation patterns driving chemotherapy resistance in ovarian high-grade serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A003.

Abstract IA012: Targeting AXL in platinum-resistant ovarian cancer: from the lab bench to Phase 3 trial

Abstract IA012: Targeting AXL in platinum-resistant ovarian cancer: from the lab bench to Phase 3 trial

Biofunctional study on chemoresistance in esophageal squamous carcinoma cells induced by missense mutation of NOTCH1 p.E450K.

Neoadjuvant chemotherapy (nCT) combined with surgery is one of the main strategies for the treatment of resectable locally advanced esophageal squamous cell carcinoma (ESCC). However, nearly 40% of patients did not benefit from nCT, and the detection rate of NOTCH1 missense mutation was significantly increased in patients who did not respond to chemotherapy, suggesting that the missense mutation may be related to tumor chemoresistance. We aim to explore the effect of a NOTCH1 missense mutation on cell phenotype, to interpret the biofunctional changes in cell lines with a NOTCH1 missense mutation and to analyze the effect of a NOTCH1 missense mutation on drug resistance in ESCC cell lines. Sanger sequencing was used to evaluate the exon mutations in the NOTCH1 ligand binding region of candidate ESCC cell lines. After screening, KYSE450 and KYSE140 cells were selected as the research objects, and point mutation cell lines [KYSE140-mutant-type (MT) and KYSE450-MT] were constructed by CRISPR/Cas9 technology. Then, functional experiments were performed with the four cell lines [KYSE450-MT/wild-type (WT) and KYSE140-MT/WT]. The drug resistance of ESCC cell lines was assessed with a drug sensitivity test, and the proliferation, invasion and migration of ESCC lines were evaluated by proliferation test, scratch test and Transwell test. The cell cycle status of ESCC cells was assessed using flow cytometry. Drug sensitivity tests showed that the NOTCH1 p.E450K point mutation caused chemotherapy resistance in KYSE140 and KYSE450 ESCC cell lines. Cell proliferation, Wound scratch and Transwell assays showed that the NOTCH1 p.E450K point mutation enhanced the proliferation, invasion and migration abilities of KYSE140 and KYSE450 cells. Flow cytometry analysis showed that the NOTCH1 p.E450K point mutation caused an increase in KYSE140 and KYSE450 cells in S phase. The NOTCH1 p.E450K point mutation causes chemotherapy resistance in KYSE140 and KYSE450 ESCC cells. Cell functional experiments showed that the NOTCH1 p.E450K point mutation enhanced the proliferation, migration and invasion abilities of KYSE140 and KYSE450 cells and increased the number of cells in S phase.

New Insights into Chemoresistance Mediated by Mdm2 Inhibitors: The Benefits of Targeted Therapy over Common Cytostatics.

The inhibition of the Mdm2-p53 protein-protein interaction is a promising strategy for anticancer therapy. However, the problem of developing secondary chemoresistance in tumors treated with such drugs has not yet been sufficiently studied. In this work, we compared the properties of a drug-resistant cell line obtained during long-term cultivation in the presence of an Mdm2 inhibitor, Nutlin-3a, with a similarly obtained line insensitive to the cytostatic drug paclitaxel. We first confirmed the higher safety levels of Mdm2 inhibitors when compared with cytostatics in terms of the development of secondary chemoresistance. We showed that Nutlin-3a affects both the targeted p53-mediated cellular machinery and the universal ABC-mediated efflux mechanism. While both targeted and general defense mechanisms are activated by the Mdm2 inhibitor, it still increases the susceptibility of tumor cells to other drugs. The results obtained indicate that the risks of developing chemoresistance under the therapy with a targeted agent are fundamentally lower than during cytotoxic therapy.

Different views of the pathway's impact on Chemoresistance and Relapse Advanced Breast Cancer and type 2 Diabetes Mellitus

Advanced breast cancer (ABC) with type 2 diabetes mellitus could be an uncommon, however profoundly forceful frame of breast cancer, which accounts for less than 5% of all locally progressed introductions. The clinical introduction of advanced breast cancer regularly contrasts altogether from that of non-advanced breast cancer; immunohistochemistry uncovers a few recognizing highlights. The more forceful triple-negative and HER2-positive breast cancer subtypes are overrepresented in advanced breast cancer compared to non-advanced breast cancer, with a poorer guess in reaction to routine treatments and plasma glucose level control with hyperglucosemia modification. Current understanding of breast tumor chemoresistance - breast cancers localized at essential breast areas and treated early can still relapse because of the presence of cancer cells and the change of cancer cells into a moderately forceful phenotype. This audit summarizes the current proof recommending that inflammatory signaling pathways are up-regulated, which may give a road for novel therapeutics against the background of diabetes. The part of the tumor microenvironment, through tumor-associated macrophages and infiltrating lymphocytes, is additionally examined, recommending that these tumors' outward variables may offer assistance in accounting for the contrasts in behavior between advanced breast cancer with high plasma glucose levels and non-advanced breast cancer. There are different novel treatment techniques currently underway in clinical trials; they require encouraging the improvement of preclinical models of this uncommon but forceful disease is vital.

Tumor-Stroma Proportion to Predict Chemoresistance in Patients With Ovarian Cancer

Platinum-based chemotherapy is the backbone of standard-of-care treatment for patients with advanced-stage, high-grade serous carcinoma (HGSC), the most common form of ovarian cancer; however, one-third of patients have or acquire chemoresistance toward platinum-based therapies. To demonstrate the utility of tumor-stroma proportion (TSP) as a predictive biomarker of chemoresistance of HGSC, progression-free survival (PFS), and overall survival (OS). This prognostic study leveraged tumors from patients with HGSC in The Cancer Genome Atlas (TCGA) cohort (1993-2013) and an independent cohort of resected clinical specimens from patients with HGSC (2004-2014) available in diagnostic and tissue microarray formats from the University of Tübingen in Germany. Data analysis was conducted from January 2021 to January 2024. Diagnosis of HGSC. Principal outcome measures were the ability of TSP to predict platinum chemoresistance, PFS, and OS. Using hematoxylin and eosin-stained slides from the Tübingen cohort (used for routine diagnostic assessment from surgical specimens) as well as tissue microarrays, representative sections of tumors for scoring of TSP were identified using previously evaluated cutoffs of 50% stroma or greater (high TSP) and less than 50% stroma (low TSP). Digitized slides from the TCGA Cohort were analyzed and scored in a similar fashion. Kaplan-Meier time-to-event functions were fit to estimate PFS and OS. The study included 103 patients (mean [SD] age, 61.6 [11.1] years) from the TCGA cohort and 192 patients (mean [SD] age at diagnosis, 63.7 [11.1] years) from the Tübingen cohort. In the TCGA cohort, there was no significant association of TSP levels with chemoresistance, PFS, or OS. However, in the Tübingen cohort, high TSP was associated with significantly shorter PFS (HR, 1.586; 95% CI, 1.093-2.302; P = .02) and OS (hazard ratio [HR], 1.867; 1.249-2.789; P = .002). Patients with chemoresistant tumors were twice as likely to have high TSP as compared to patients with chemosensitive tumors (HR, 2.861; 95% CI, 1.256-6.515; P = .01). In tissue microarrays from 185 patients from the Tübingen cohort, high TSP was again associated with significantly shorter PFS (HR, 1.675; 95% CI, 1.012-2.772 P = .04) and OS (HR, 2.491; 95% CI, 1.585-3.912; P < .001). In this prognostic study, TSP was a consistent and reproducible marker of clinical outcome measures of HGSC, including PFS, OS, and platinum chemoresistance. Accurate and cost-effective predictive biomarkers of platinum chemotherapy resistance are needed to identify patients most likely to benefit from standard treatments, and TSP can easily be implemented and integrated into prospective clinical trial design and adapted to identify patients who are least likely to benefit long-term from conventional platinum-based cytotoxic chemotherapy treatment at the time of initial diagnosis.

Ultrasound-mediated intra-/extracellular dual intervening effect combined with all-trans retinoic acid for cancer stemness inhibition

Cancer stem cells (CSCs), characterized by robust self-renewal capacity and varied differentiation potential, are considered pivotal in the ineffectiveness of cancer therapy. Some strategies targeting CSCs may exhibit limited efficacy due to the rigid extracellular matrix (ECM) in tumor region that still maintains stemness through mechanosensitive pathways. Herein, an ultrasound (US)-mediated intra/extracellular dual intervening strategy was developed for effective tumor stemness inhibition. Specifically, a kind of US-responsive nanodroplets (PAP@Lipid) co-loaded with all-trans retinoic acid (ARTA) and paclitaxel (PTX) are constructed via one step emulsification process. Thereinto, the encapsulated ARTA is beneficial to CSC stemness and chemoresistance, and the perfluorohexane (PFH) core of nanodroplets enables local US enhancement through acoustic droplet vaporization (ADV) effect. Moreover, the enhanced US effect can initiate reactive oxygen species (ROS) generation and aldehyde dehydrogenase (ALDH) downregulation, synergistically diminishing CSC populations in combination with ARTA. More importantly, it also simultaneously destroys the ECM, rendering a softened tumor with reduced stemness level and enhanced vulnerability to PTX, thereby promoting therapeutic effect. The in vivo antitumor evaluations demonstrate the remarkable inhibiting effect of This US-mediated intra/extracellular intervening strategy on both 4T1 and chemoresistant tumors, providing an encouraging paradigm for combating refractory cancers.

Cancer Drug Resistance: Targeting Proliferation or Programmed Cell Death.

The development of resistance to chemotherapy is one of the main problems for effective cancer treatment. Drug resistance may result from disturbances in two important physiological processes-cell proliferation and cell death. Importantly, both processes characterize alterations in cell metabolism, the level of which is often measured using MTT/MTS assays. To examine resistance to chemotherapy, different cancer cell lines are usually used for the in vitro modulation of developing resistance. However, after the creation of resistant cell lines, researchers often have difficulty in starting investigations of the mechanisms of insensitivity. In the first stage, researchers should address the question of whether the drug resistance results from a depression of cell proliferation or an inhibition of cell death. To simplify the choice of research strategy, we have suggested a combination of different approaches which reveal the actual mechanism. This combination includes rapid and high-throughput methods such as the MTS test, the LIVE/DEAD assay, real-time cell metabolic analysis, and Western blotting. To create chemoresistant tumor cells, we used four different cancer cell lines of various origins and utilized the most clinically relevant pulse-selection approach. Applying a set of methodological approaches, we demonstrated that three of them were more capable of modulating proliferation to avoid the cytostatic effects of anti-cancer drugs. At the same time, one of the studied cell lines developed resistance to cell death, overcoming the cytotoxic action.

Intratumoral Heterogeneity, Chemoresistance and Lymph Node Landing Zone Prognosis in Testicular Tumors Based on Histopathological Characteristics.

Existing data on the histopathological correlation of testicular tumors with lymph node prognosis have been poorly explored. We aimed to investigate the relationship of the histopathological properties of testicular tumors with lymph nodes and their involvement with chemoresistance and heterogeneity of testicular tumors. Patients with non-seminomatous germ cell tumor (NSGCT) were selected for histopathological correlation of testicular tumor with lymph nodes and its relationship with chemoresistance and heterogeneity. Histopathological and radiological parameters associated with the risk of chemoresistance and tumor progression were measured pre- and post-chemotherapy. Binomial logistic regression and Kaplan-Meier analysis were implemented to determine the predictors of progression and adverse overall patient survival. All categorical variables were analyzed using the Chi-square test, while Pearson's R coefficient determined the correlation. Male patients who were diagnosed with NSGCT from March 2017 to December 2018 at Guwahati Medical College, Guwahati, India, were included in this study. Lymph node groups were predominantly incriminated with the EYST or EYS groups and minimally linked with the pure E and YCS groups. Furthermore, the highest number of lymph node stations was associated with pre-chemotherapy. In salvage chemotherapy in the form of VIP, we found exciting outcomes, as approximately 41% of cases responded positively, especially in the EYS group. Our study classifies NSGCT according to the most favorable histopathological grouping and explores the tumoral response in different intrinsic and extrinsic variables. Our analysis can serve as a triumphant histopathological nomogram for a sublime management protocol to deal with the onerous histological pairing in NSGCT.

Use of microRNAs as Diagnostic, Prognostic, and Therapeutic Tools for Glioblastoma.

Glioblastoma (GB) is the most aggressive and common type of cancer within the central nervous system (CNS). Despite the vast knowledge of its physiopathology and histology, its etiology at the molecular level has not been completely understood. Thus, attaining a cure has not been possible yet and it remains one of the deadliest types of cancer. Usually, GB is diagnosed when some symptoms have already been presented by the patient. This diagnosis is commonly based on a physical exam and imaging studies, such as computed tomography (CT) and magnetic resonance imaging (MRI), together with or followed by a surgical biopsy. As these diagnostic procedures are very invasive and often result only in the confirmation of GB presence, it is necessary to develop less invasive diagnostic and prognostic tools that lead to earlier treatment to increase GB patients' quality of life. Therefore, blood-based biomarkers (BBBs) represent excellent candidates in this context. microRNAs (miRNAs) are small, non-coding RNAs that have been demonstrated to be very stable in almost all body fluids, including saliva, serum, plasma, urine, cerebrospinal fluid (CFS), semen, and breast milk. In addition, serum-circulating and exosome-contained miRNAs have been successfully used to better classify subtypes of cancer at the molecular level and make better choices regarding the best treatment for specific cases. Moreover, as miRNAs regulate multiple target genes and can also act as tumor suppressors and oncogenes, they are involved in the appearance, progression, and even chemoresistance of most tumors. Thus, in this review, we discuss how dysregulated miRNAs in GB can be used as early diagnosis and prognosis biomarkers as well as molecular markers to subclassify GB cases and provide more personalized treatments, which may have a better response against GB. In addition, we discuss the therapeutic potential of miRNAs, the current challenges to their clinical application, and future directions in the field.

FSP1 is a predictive biomarker of osteosarcoma cells’ susceptibility to ferroptotic cell death and a potential therapeutic target

Human osteosarcoma (OS) is a relatively rare malignancy preferentially affecting long body bones which prognosis is often poor also due to the lack of effective therapies. Clinical management of this cancer basically relies on surgical removal of primary tumor coupled with radio/chemotherapy. Unfortunately, most osteosarcoma cells are resistant to conventional therapy, with the undergoing epithelial-mesenchymal transition (EMT) giving rise to gene expression reprogramming, thus increasing cancer cell invasiveness and metastatic potential. Alternative clinical approaches are thus urgently needed. In this context, the recently described ferroptotic cell death represents an attractive new strategy to efficiently kill cancer cells, since most chemoresistant and mesenchymal-shaped tumors display high susceptibility to pro-ferroptotic compounds. However, cancer cells have also evolved anti-ferroptotic strategies, which somehow sustain their survival upon ferroptosis induction. Indeed, here we show that osteosarcoma cell lines display heterogeneous sensitivity to ferroptosis execution, correlating with the mesenchymal phenotype, which is consistently affected by the expression of the well-known anti-ferroptotic factor ferroptosis suppressor protein 1 (FSP1). Interestingly, inhibiting the activity or expression of FSP1 restores cancer cell sensitivity to ferroptosis. Moreover, we also found that: i) AKRs might also contribute to resistance; ii) NRF2 enhances FSP1 expression upon ferroptosis induction; while iii) p53 contributes to the regulation of FSP1 basal expression in OS cells.In conclusion, FSP1 expression can potentially be used as a valuable predictive marker of OS sensitivity to ferroptosis and as a new potential therapeutic target.

Exploring Tumor-Promoting Qualities of Cancer-Associated Fibroblasts and Innovative Drug Discovery Strategies With Emphasis on Thymoquinone.

Tumor epithelial development and chemoresistance are highly promoted by the tumor microenvironment (TME), which is mostly made up of the cancer stroma. This is due to several causes. Cancer-associated fibroblasts (CAFs) stand out among them as being essential for the promotion of tumors. Understanding the fibroblastic population within a single tumor is made more challenging by the undeniable heterogeneity within it, even though particular stromal alterations are still up for debate. Numerous chemical signals released by tumors improve the connections between heterotypic fibroblasts and CAFs, promoting the spread of cancer. It becomes essential to have a thorough understanding of this complex microenvironment to effectively prevent solid tumor growth. Important new insights into the role of CAFs in the TME have been revealed by recent studies. The objective of this review is to carefully investigate the relationship between CAFs in tumors and plant secondary metabolites, with a focus on thymoquinone (TQ). The literature published between 2010 and 2023 was searched in PubMed and Google Scholar with keywords such as TQ, TME, cancer-associated fibroblasts, mechanism of action, and flavonoids. The results showed a wealth of data substantiating the activity of plant secondary metabolites, particularly TQ's involvement in blocking CAF operations. Scrutinized research also clarified the wider effect of flavonoids on pathways related to cancer. The present study highlights the complex dynamics of the TME and emphasizes the critical role of CAFs. It also examines the possible interventions provided by secondary metabolites found in plants, with TQ playing a vital role in regulating CAF function based on recent literature.

Microglia-mediated drug substance transfer promotes chemoresistance in brain tumors: insights from an in vitro co-culture model using GCV/Tk prodrug system

BackgroundIt is well known that tumor-associated macrophages (TAMs) play essential roles in brain tumor resistance to chemotherapy. However, the detailed mechanisms of how TAMs are involved in brain tumor resistance are still unclear and lack a suitable analysis model.MethodsA BV2 microglial cells with ALTS1C1 astrocytoma cells in vitro co-culture system was used to mimic the microglia dominating tumor stroma in the tumor invasion microenvironment and explore the interaction between microglia and brain tumor cells.ResultsOur result suggested that microglia could form colonies with glioma cells under high-density culturing conditions and protect glioma cells from apoptosis induced by chemotherapeutic drugs. Moreover, this study demonstrates that microglia could hijack drug substances from the glioma cells and reduce the drug intensity of ALTS1C1 via direct contact. Inhibition of gap junction protein prevented microglial-glioma colony formation and microglia-mediated chemoresistance.ConclusionsThis study provides novel insights into how glioma cells acquire chemoresistance via microglia-mediated drug substance transferring, providing a new option for treating chemo-resistant brain tumors.

Gastric cancer mesenchymal stem cells promote tumor glycolysis and chemoresistance by regulating B7H3 in gastric cancer cells.

Despite surgical treatment combined with multidrug therapy having made some progress, chemotherapy resistance is the main cause of recurrence and death of gastric cancer (GC).Gastric cancer mesenchymal stem cells (GCMSCs)have been reported to be correlated with the limited efficacy of chemotherapy in GC, but the mechanism of GCMSCs regulating GC resistance needs to be further studied. The gene set enrichment analysis (GSEA)was performed to explore the glycolysis-related pathways heterogeneity across different cell subpopulations. Glucose uptake and lactate production assays were used to evaluate the importance of B7H3 expression in GCMSCs-treated GCcells. The therapeutic efficacy of oxaliplatin (OXA)and paclitaxel (PTX)was determined using CCK-8and colony formation assays. Signaling pathways altered by GCMSCs-CMwere revealed by immunoblotting. The expression of TNF-αin GCMSCs and bone marrow mesenchymal stem cells (BMMSCs)was detected by western blot analysis and qPCR. Our results showed that the OXA and PTX resistance of GC cells were significantly enhanced in the GCMSCs-CMtreated GC cells. Acquired OXA and PTX resistance was characterized by increased cell viability for OXA and PTX, the formation of cell colonies, and decreased levels of cell apoptosis, which were accompanied by reduced levels of cleaved caspase-3 and Bax expression, and increased levels of Bcl-2, HK2, MDR1, and B7H3 expression. Blocking TNF-αin GCMSCs-CM,B7H3 knockdown or the use of 2-DG,a key enzyme inhibitor of glycolysis in GC cells suppressed the OXA and PTX resistance of GC cells that had been treated with GCMSCs-CM. This study shows that GCMSCs-CMderived TNF-α could upregulate the expression of B7H3 of GC cells to promote tumor chemoresistance. Our results provide a new basis for the treatment of GC.

Abstract B062: CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to therapy. Stromal cancer-associated fibroblasts (CAFs) play a key role in promoting tumor progression and chemoresistance, by creating a fibrotic microenvironment that impedes drug access and feeds PDAC cells nutrients and pro-tumor signals. CRO-67 (Patent PCT/AU2023/050505) is a novel drug developed with Noxopharm Ltd using a rational medicinal chemistry design to improve bioavailability of chromans which have potent anti-cancer activity. Using our patient-derived PDAC tumor in a dish model (explants; maintain multicellular architecture and fibrosis) we previously showed that CRO-67 has both anti-tumor and CAF reprogramming capacity in 4 patient explants [Cancer Res (2022) 82 (22_Supplement):C073]. Aims: 1) To expand evaluation of CRO-67 in additional patient-derived PDAC tumor explants given PDAC heterogeneity. 2) Assess the effect of CRO-67 on PDAC cell and CAF function. 3) Validate the therapeutic potential of CRO-67 on PDAC growth in vivo. Methods: 1) PDAC tumor samples collected from 7 patients undergoing pancreatic resection. Tumor explants (1–2mm diameter) were cultured on gelatin sponges, treated with CRO-67 (0–50μg/mL) every 3 days and fixed on day 12. Therapeutic response assessed by immunohistochemistry for cytokeratin (PDAC cells), α-smooth muscle actin (CAFs), bromodeoxyuridine (proliferation) and TUNEL (cell death). 2) PDAC cells (MiaPaCa-2) and patient-derived CAFs were treated with CRO-67 (1.5μM) for 48h before proliferation analysis (IncuCYTE S3) and for 24h before apoptosis (Annexin V/DAPI staining; flow cytometry) and cell cycle (DAPI staining; flow cytometry) analysis. 3) Subcutaneous PDAC (BxPC3 human cells) tumors in mice were treated with CRO-67 (2.5mg/kg Intraperitoneally, twice a day) for 21 days and tumor volume measured (calipers). Results: 1) CRO-67 treatment decreased tumor and CAF cell frequency in 6/6 (no quantifiable tumor in patient 7 explants) and 7/7 patient PDAC explants, respectively. It also decreased explant cell proliferation and increased cell death versus controls. 2) CRO-67 completely abolished proliferation of MiaPaCa-2 and reduced the average growth rate of CAFs by 81.3±8.8% (p=0.0025; n=5), increased apoptosis in MiaPaCa-2 by 455.7±21.4% (p=0.003; n=3) and in CAFs by 172.6±13.1% (p=0.0067; n=5) and increased the fraction of cells in G2/M cell cycle phase by 163.8±12.8% (p&amp;lt;0.0001; n=3) in MiaPaCa-2 and by 74.6±8.6% (p=0.0015; n=5) in CAFs versus controls. 3) CRO-67 reduced PDAC tumors in vivo by 56.7±6.6% (p=0.0013; n=9 mice/group) versus controls. Conclusions: CRO-67: 1) reduced PDAC cells and CAFs in human PDAC explants via reduced proliferation and increased death; 2) reduced proliferation of PDAC cells and CAFs in vitro, increased apoptosis and disrupted cell cycle progression through G2/M phase; 3) reduced PDAC tumor growth in vivo. Implication: We predict CRO-67 is a potential ‘dual cell’ therapy with direct anti-tumor effects and CAF reprogramming capacity, warranting further investigation in vivo. Citation Format: Keilah Garcia Netto, Shannon Chiang, John Kokkinos, Koroush S. Haghighi, Aparna Raina, Omali Pitiyarachchi, Janet Youkhana, Quach Truong, Daniel Wenholz, Xiang Li, Olivier Laczka, Naresh Kumar, John Wilkinson, David Goldstein, George Sharbeen, Phoebe A. Phillips. CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B062.