Hormone receptor-positive (HR+) and HER2-negative (HER2−) breast cancer represents ~70% of breast tumors. Accurate detection of PIK3CA and ESR1 mutations is crucial for guiding targeted therapies, particularly in advanced disease. Liquid biopsy testing is increasingly recommended, especially for ESR1 mutation, but standardized genotyping methods remain limited. The CANIPE study, a prospective, multicenter Spanish trial, evaluated mutation prevalence in 106 baseline blood samples. PIK3CA mutations were identified in 41.9% by ddPCR droplet digital PCR (ddPCR) and 42.8% by targeted next-generation sequencing (NGS), while ESR1 mutations were detected in 7.8 and 9.5%, respectively. A concordance analysis between ddPCR and NGS was performed in 62 paired samples, demonstrating excellent agreement (Kappa >0.85) and strong correlation in variant allele frequency for PIK3CA, confirming the reliability of the methods. Baseline circulating free DNA levels were prognostic for survival. These results, derived from real-world patient samples, support ddPCR and NGS as robust, complementary tools for mutation detection and therapeutic decision-making.

Approximatly 70% of breast cancer (BC) patients are Hormone Receptors (HR) positive, the most common subtype with the best prognosis. The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with Endocrine Therapy (ET) is tje standard firs-line therapy for HR+/HER2- metastatic BC. However, 20% of patients are intrinsically resistant, and those who initially respond often develop acquired resistance, making the management of resistant HR+/HER2- metastatic BC a significant clinical challenge. Clinical guidelines emphasize the need of comprehensive real-time monitoring of the dynamic mutational landscape during and after treatment to improve resistance prediction. Early detection of mutations in ESR1 and the PIK3 pathway usinf circulating tumor DNA (ctDNA) may allow the ending of ineffective endocrine therapies and initiation of alternative treatments for these patients, without performing tissue biopsies before radiological progression occurs. To achieve this, it is crucial to implement non-onvasive genotyping that allows better-adapted pharmacologiical interventions. However, clinical trials have been predominantly carried out with selected populations and single drugs (Palbociclib, Ribociclib or Abemaciclib). There is a lack of studies of optimal agreemnt between diagnostic methods, especially in liquid biopsy. There is no relieble data abaout PIK3CA or ESR1 status in ctDNA from real-world cohorts that could help to define the best testing strategy for the clinica routine. Therefore, it is imperative to establish evidence that the use of ddPCR is equally or more sensitive for ctDNA analysis than qPCR or NGS, the current gold standard methods. Aim: This study aims to perform a concordance analysis between NGS and ddPCR technologies for detecting mutations in PIK3CA and ESR1 by testing the ctDNA from HR+/HER2- metastatic breast cancer patients. M&M: CANIPE is a randomised, open-label study performed al 14 Spanish hospitals in 6 autonomous communities. Patients aged 18 years or older, with confirmed HR+/HER2- breast cancer, stage IV, Eastern Cooperative Oncology Group performance status (ECOG-PS) of 0 to 2, who iniciate first-line tratment with CDK4/6i plus aromatase inhibitors. All procedures followed the Helsinki Declaration guidelines and were approved by the Ethics Commitee of Santiago-Lugo under approval reference number 2022/386. All patients provide written informed consent. A total of 60 women were recruited at baseline (before therapy initiation) while 21 patients at 10 months after treatment. 40 mL of blood was obteined from each patient at both time points. For ddPCR, cfDNA was isolated from 5 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Venlo, The Netherlands). Multiplex ddPCR was performed with de Bio-Rad QX-200 system to identify PIK3CA mutations, including the most frequent variants (E542K, E545K, H1047L, H1047R, R88Q, N345K, C420R, E545A, E545G,Q546K), and ESR1 mutations (L536R, D538G, E380Q, Y537C, /537S, Y537N,S463P). A mutation was considered present if at least 6 mutated events were detected by ddPCR. Additionally, for NGS, cfDNA was isolated from 4 mL of plasma and analysed with the AVENIO ctDNA Expanded kit (Roche Diagnostics), which includes 77 genes, including PIK3CA and ESR1. An allele fraction od 0.5 % or higher was consideredindicative of a mutation. Results: At baseline, and considering the pre-defined criteria, ctDNA analysis detected PIK3CA mutations in 32.25% and 44.44% of patients by AVENIO and ddPCR, respectively. ESR1 mutations were detected in 3.22% and 9.37% by AVENIO and ddPCR, respectively. For PIK3CA mutations, the Kappa value was 0.62 (p-value: 0.0004) and 0.47 for ESR1 mutations (p-value: 0.0039). However, when NGS data was included for mutations with allele fraction less than 0.5%, the Kappa value was 0.92 for PIK3CA and 1 for ESR1, indicating almost perfect agreement bvetween the two technologies (p-value < 0.0001). Conclusion: The analysis of ctDNA bu Multiplex ddPCR of HR+/HER2- metastatic breas cancer patients represent a sensitive tool to identify PIK3CA and ESR1 mutations with a high concordance compared with the NGS, which is currently the reference technology. Citation Format: Teresa Curiel, Carmela Rodriguez, Aitor Rodriguez Casanova, Nerea González, Ramón Lago-Lestón, Martín Giráldez, Alicia Abalo, Carmen Abuín, Maribel Aibar, Patricia Palacios, Juan Cueva, Marta carmona, Alexia Cortegoso, Serafín Morales, Josep Gumá, Mariana López Flores, Yolanda Fernández, Isaura Fernádez, Ignacio Fernández, María Gión, Carolina Pena, Jesús García Mata, Andrea Saenz de Miera, Angel Díaz Lagares, Laura Muinelo Romay, Clotilde Costa, Rafael López López. Concordance Analysis of Non-Invasive determination techniques of PIK3CA and ESR1 mutations in patients with advanced luminal breast cancer. Study CANIPE [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-02-17.

A critical step in the metastatic cascade is the survival of circulating tumor cells (CTCs) within the bloodstream. Although interactions between CTCs and various hematopoietic cells have been described, the role of red blood cells (RBCs) remains underexplored. This study investigated the interactions between tumor cells and RBCs from breast and lung cancer patients, revealing significant phenotypic and functional changes in tumor cells, unlike interactions with RBCs from healthy donors. Tumor cell and patient-derived RBC co-cultures increased tumor cell attachment and induced morphological changes. RBC-primed tumor cells showed increased adhesion, disruption of the endothelial barrier, and invasiveness, both in vitro and in vivo. Global proteome changes, including actin remodeling and VASP accumulation at cell edges, promote directional migration. RBCs from patients with metastatic breast cancer also upregulate PAK4, enhancing migration and epithelial-mesenchymal transition, whereas PAK4 inhibition reduces these effects. Clinically, a higher red blood cell distribution width (RDW) in patients with metastasis is associated with increased CTC counts and poor outcomes. This study highlights the previously unrecognized role of RBCs in promoting metastatic behavior in cancer cells and suggests potential therapeutic targets, such as PAK4, to counteract these effects.

Metastatic breast cancer (BC) is the main cause of cancer-related mortality in women worldwide. HR + /HER2- BC patients are treated with endocrine therapy (ET), but therapeutic resistance is common. The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with ET was approved for metastatic BC patients and extended the median progression-free survival to 24 months. This therapy is not always effective, and in every patient, resistance ultimately occurs, but the underlying resistance mechanisms remain unclear. To address this gap, we explored circulating tumour cells (CTCs) as biomarkers to assess treatment response and resistance in metastatic HR + /HER2- BC patients receiving CDK4/6i plus ET. In total, 53 HR + /HER2- metastatic BC patients who received a CDK4/6i plus ET as first-line treatment were analysed, including samples from internal and external validation cohorts. CTCs were isolated using the negative enrichment approach RosetteSep (STEMCELL Technologies) or positive immunomagnetic selection targeting EpCAM, EGFR, and HER2 (AdnaTest EMT-2/StemCell Select™, QIAGEN). RNA was extracted from CTCs and PBMCs for nCounter analysis (Pancancer pathways panel) in a discovery phase. Subsequent validation was performed by RT-qPCR. CTC gene expression analysis revealed that non responder patients (those who experienced disease progression before 180 days) exhibited elevated PRKCB (p-value: 0.011), MAPK3 (p-value: 0.006) and STAT3 (p-value: 0.008) expression, while responders showed increased CDK6 (p-value: 0.011) and CCND1 (p-value: 0.035) expression at baseline. CTC transcriptional characterization revealed a gene expression signature (STAT3highPRKCBhighCDK6low) that accurately classified HR + /HER2- metastatic BC patients who responded to CDK4/6i plus ET, regardless of the CTC isolation method (AUC > 0.8). CTC characterization at progression also identified biomarkers linked to therapy resistance, including the epigenetic regulators EZH2 and HDAC6 and the cell cycle regulator CDC7, which could guide the selection of subsequent therapy lines. The expression of the CDK4 and STAT3 genes in CTCs was associated with progression-free survival and overall survival, respectively. Likewise, the presence of ≥ one CTC after one cycle of therapy predicts a worse prognosis. CTC gene expression provides information about treatment outcomes in HR + /HER2- metastatic BC patients receiving CDK4/6i plus ET and could guide personalized strategies and improve prognosis.

Background: Circulating tumor cells (CTCs) and CTC-clusters are pivotal in the metastatic process of breast cancer (BC). Owing to their low frequency, models replicating their biology should provide a robust platform for investigating the molecular mechanisms driving metastasis and identifying new biomarkers. We established and characterized a CTC-derived cell model from a mouse xenograft to explore its metastatic behavior and molecular profile, which allowed us to investigate the expression and prognostic significance of a set of genes associated with the metastatic potential of CTCs. Methods: The CTC line (mCTC) derived from a MDA-MB-231 mouse xenografts was used in comparative functional analyses including cell cycle evaluation, colony formation, invasion, adhesion, and metastatic competency in zebrafish models. Transcriptomic profiling and functional assays were conducted to identify candidate genes and understand their roles in metastasis. Moreover, publicly available gene expression datasets of CTCs, CTC-clusters, and tumor tissue, from GEO and TCGA, were analyzed for the identification of a gene signature that was correlated with survival data. The signature was validated in an independent cohort. Results: Compared with MDA-MB-231 cells, mCTC cells presented enhanced colony formation, invasion, and adhesion, and increased dissemination and survival in zebrafish. Transcriptomic analysis revealed that SPARC was significantly upregulated. Functional assays showed that SPARC overexpression was correlated with increased invasion and migration. Analysis of public datasets confirmed the high expression of SPARC in BC CTCs and CTC-clusters. Additionally, a 4-gene signature involving SPARC, THBS1, VCL, and HSP90AB1 was identified that demonstrated strong prognostic value, predicting shorter overall and distant metastasis-free survival in the primary tumor setting. Validation cohorts confirmed its ability to distinguish high-risk patients. Elevated expression of the 4-gene signature in CTCs was also indicative of increased mortality risk. Conclusion: mCTC exhibit distinct metastatic traits and molecular characteristics, highlighting a possible role of SPARC in CTC biology and its potential as a prognostic marker in BC metastasis. The identified 4-gene signature provides a robust prognostic tool for assessing patient risk and guiding therapeutic strategies. Further investigations into the mechanistic role of SPARC may reveal new therapeutic targets for managing BC progression.

One critical step in the metastatic cascade is the survival of circulating tumour cells (CTCs) within the bloodstream. While numerous interactions between CTCs and various hematopoietic cells have been described, the role of red blood cells (RBCs) in this process remains underexplored. This study investigates the interactions between tumour cells and RBCs from breast and lung cancer patients, revealing significant phenotypic and functional changes in the tumour cells, unlike when the contact is with RBCs from healthy donors. In vitro co-culture of cancer cell lines with RBCs from metastatic cancer patients resulted in increased tumour cell attachment accompanied by morphological changes. Additionally, RBCs-primed tumour cells showed increased adhesion and disruption of the endothelial barrier in vitro and increased invasiveness both in vitro and in vivo. Transcriptomic analysis showed that RBCs from metastatic breast cancer patients induce significant gene expression changes, notably upregulating PAK4, which enhances migration and epithelial-mesenchymal transition. PAK4 inhibition reduced these effects. Proteomic studies revealed substantial remodelling, including actin-related changes and the accumulation of VASP at cell edges, promoting directional migration. Clinically, higher RBC distribution width (RDW) in metastatic breast cancer patients is associated with increased CTC counts and worse outcomes. This study highlights the previously unrecognized role of RBCs in promoting metastatic behaviours in cancer cells and suggests potential therapeutic targets, such as PAK4, to counteract these effects. Further exploration of RBCs-tumour cell interactions could provide new insights into metastatic mechanisms and improve cancer prognosis and treatment strategies. ### Competing Interest Statement The authors have declared no competing interest.

The dynamic intercommunication between tumour cells and cells from the microenvironment, such as cancer-associated fibroblast (CAFs), is a key factor driving breast cancer (BC) metastasis. Clusters of circulating tumour cells (CTCs), known to bare a higher efficiency at establishing metastases, are found in the blood of BC patients, often accompanied by CAFs in heterotypic CTC-clusters. Previously we have shown the utility of CTC-clusters models and the zebrafish embryo as a model of metastasis to understand the biology of breast cancer CTC-clusters. In this work, we use the zebrafish embryo to study the interactions between CTCs in homotypic clusters and CTC-CAFs in heterotypic CTC-clusters to identify potential pro-metastatic traits derived from CTC-CAF communication. We found that upon dissemination CAFs seem to exert a pro-survival and pro-proliferative effect on the CTCs, but only when CTCs and CAFs remain joined as cell clusters. Our data indicate that the clustering of CTC and CAF allows the establishment of physical interactions that when maintained over time favour the selection of CTCs with a higher capacity to survive and proliferate upon dissemination. Importantly, this effect seems to be dependent on the survival of disseminated CAFs and was not observed in the presence of normal fibroblasts. Moreover, we show that CAFs can exert regulatory effects on the CTCs without being involved in promoting tumour cell invasion. Lastly, we show that the physical communication between BC cells and CAFs leads to the production of soluble factors involved in BC cell survival and proliferation. These findings suggest the existence of a CAF-regulatory effect on CTC survival and proliferation sustained by cell-to-cell contacts and highlight the need to understand the molecular mechanisms that mediate the interaction between the CTCs and CAFs in clusters enhancing the metastatic capacity of CTCs.

Breast cancers of the luminal B subtype are frequent tumors with high proliferation and poor prognosis. Epigenetic alterations have been found in breast tumors and in biological fluids. We aimed to profile the cell-free DNA (cfDNA) methylome of metastatic luminal B breast cancer (LBBC) patients using an epigenomic approach to discover potential noninvasive biomarkers. Plasma cfDNA was analyzed using the Infinium MethylationEpic array in a cohort of 14 women, including metastatic LBBC patients and nontumor controls. The methylation levels of cfDNA and tissue samples were validated with droplet digital PCR. The methylation and gene expression data of 582 primary luminal breast tumors and 79 nontumor tissues were obtained from The Cancer Genome Atlas (TCGA). We found an episignature of 1,467 differentially methylated CpGs that clearly identified patients with LBBC. Among the genes identified, the promoter hypermethylation of WNT1 was validated in cfDNA, showing an area under the ROC curve (AUC) of 0.86 for the noninvasive detection of metastatic LBBC. Both paired cfDNA and primary/metastatic breast tumor samples showed hypermethylation of WNT1. TCGA analysis revealed significant WNT1 hypermethylation in the primary tumors of luminal breast cancer patients, with a negative association between WNT1 methylation and gene expression. In this proof-of-principle study, we discovered an episignature associated with metastatic LBBC using a genome-wide cfDNA methylation approach. We also identified the promoter hypermethylation of WNT1 in cfDNA as a potential noninvasive biomarker for luminal breast cancer. Our results support the use of EPIC arrays to identify new epigenetic noninvasive biomarkers in breast cancer.

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults' RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.

Circulating Tumor Cells (CTCs) are shed from primary tumors and travel through the blood, generating metastases. CTCs represents a useful tool to understand the biology of metastasis in cancer disease. However, there is a lack of standardized protocols to isolate and culture them. In our previous work, we presented oil-in-water nanoemulsions (NEs) composed of lipids and fatty acids, which showed a benefit in supporting CTC cultures from metastatic breast cancer patients. Here, we present Peptide-Functionalized Nanoemulsions (Pept-NEs), with the aim of using them as a tool for CTC isolation and culture in situ. Therefore, NEs from our previous work were surface-decorated with the peptides Pep10 and GE11, which act as ligands towards the specific cell membrane proteins EpCAM and EGFR, respectively. We selected the best surface to deposit a layer of these Pept-NEs through a Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) method. Next, we validated the specific recognition of Pept-NEs for their protein targets EpCAM and EGFR by QCM-D and fluorescence microscopy. Finally, a layer of Pept-NEs was deposited in a culture well-plate, and cells were cultured on for 9 days in order to confirm the feasibility of the Pept-NEs as a cell growth support. This work presents peptide-functionalized nanoemulsions as a basis for the development of devices for the isolation and culture of CTCs in situ due to their ability to specifically interact with membrane proteins expressed in CTCs, and because cells are capable of growing on top of them.

BACKGROUND:Circulating tumor cells (CTC) have relevance as prognostic markers in breast cancer. However, the functional properties of CTCs or their molecular characterization have not been well-studied. Experimental models indicate that only a few cells can survive in the circulation and eventually metastasize. Thus, it is essential to identify these surviving cells capable of forming such metastases.METHODS:We isolated viable CTCs from 50 peripheral blood samples obtained from 35 patients with advanced metastatic breast cancer using RosetteSepTM for ex vivo culture. The CTCs were seeded and monitored on plates under low adherence conditions and with media supplemented with growth factors and Nanoemulsions. Phenotypic analysis was performed by immunofluorescence and gene expression analysis using RT-PCR and CTCs counting by the Cellsearch® system.RESULTS:We found that in 75% of samples the CTC cultures lasted more than 23 days, predicting a shorter Progression-Free Survival in these patients, independently of having ≥5 CTC by Cellsearch®. We also observed that CTCs before and after culture showed a different gene expression profile.CONCLUSIONS:the cultivability of CTCs is a predictive factor. Furthermore, the subset of cells capable of growing ex vivo show stem or mesenchymal features and may represent the CTC population with metastatic potential in vivo.

BACKGROUND:Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear.METHODS:Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms.RESULTS:CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes.CONCLUSIONS:The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters.

CTCs have extensively been used for the monitoring and characterization of metastatic prostate cancer, but their application in the clinic is still very scarce. Besides, the resistance mechanisms linked to prostate cancer treatment remain unclear. Liquid biopsies represent the most promising alternative due to the complexity of biopsying bone metastasis and the duration of the disease. We performed a prospective longitudinal study in CTCs from 20 castration-resistant prostate cancer patients treated with docetaxel. For that, we used CellSearch® technology and a custom gene expression panel with qRT-PCR using a CTCs negative enrichment approach. We found that CTCs showed a hybrid phenotype during the disease, where epithelial features were associated with the presence of ≥ 5 CTCs/7.5 mL of blood, while high relative expression of the gene MYCL was observed preferentially in the set of samples with < 5 CTCs/7.5 mL of blood. At baseline, patients whose CTCs had stem or hybrid features showed a later progression. After 1 cycle of docetaxel, high relative expression of ZEB1 indicated worse outcome, while KRT19 and KLK3 high expression could predisposed the patients to a worse prognosis at clinical progression. In the present work we describe biomarkers with clinical relevance for the prediction of early response or resistance in castration-resistant prostate cancer patients. Besides, we question the utility of targeted isolated CTCs and the use of a limited number of markers to define the CTCs population.

Circulating tumor cell (CTC) enumeration has emerged as a powerful biomarker for the assessment of prognosis and the response to treatment in metastatic breast cancer (MBC). Moreover, clinical evidences show that CTC-cluster counts add prognostic information to CTC enumeration, however, their significance is not well understood, and more clinical evidences are needed. We aim to evaluate the prognostic value of longitudinally collected single CTCs and CTC-clusters in a heterogeneous real-world cohort of 54 MBC patients. Blood samples were longitudinally collected at baseline and follow up. CTC and CTC-cluster enumeration was performed using the CellSearch® system. Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Cox proportional hazards modelling. Elevated CTC counts and CTC-clusters at baseline were significantly associated with a shorter survival time. In joint analysis, patients with high CTC counts and CTC-cluster at baseline were at a higher risk of progression and death, and longitudinal analysis showed that patients with CTC-clusters had significantly shorter survival compared to patients without clusters. Moreover, patients with CTC-cluster of a larger size were at a higher risk of death. A longitudinal analysis of a real-world cohort of MBC patients indicates that CTC-clusters analysis provides additional prognostic value to single CTC enumeration, and that CTC-cluster size correlates with patient outcome.

The study of circulating tumor cells (CTCs) has a huge clinical interest in advance and metastatic breast cancer patients. However, many approaches are biased by the use of epithelial markers, which underestimate non-epithelial CTCs phenotypes. CTCs enumeration provides valuable prognostic information; however, molecular characterization could be the best option to monitor patients throughout the disease since it may provide more relevant clinical information to the physicians. In this work, we aimed at enumerating and performing a molecular characterization of CTCs from a cohort of 20 patients with metastatic breast cancer (MBC), monitoring the disease at different time points of the therapy, and at progression when it occurred. To this end, we used a CTC negative enrichment protocol that allowed us to recover a higher variety of CTCs phenotypes. With this strategy, we were able to obtain gene expression data from CTCs from all the patients. In addition, we found that high expression levels of PALB2 and MYC were associated with a worse outcome. Interestingly, we identified that CTCs with an EpCAMhighVIMlowALDH1A1high signature showed both shorter overall survival (OS) and progression-free survival (PFS), suggesting that CTCs with epithelial-stem features had the most aggressive phenotype.

Tumor growth and metastasis entangle the alteration and recruitment of non-malignant cells to the primary tumor, among them immune cells, constituting the tumor microenvironment (TME). Communication between tumor cells and their stroma has been shown as a fundamental driving force of the tumoral process. A great deal of effort has been focused on depicting their specific interactions and crosstalk. However, most research has been carried out in 2D conventional cultures that alter cell morphology and intracellular signaling processes. Considering these premises, we have developed a 3D cell co-culture model to mimic T cell infiltration into the tumor mass and explore tumor-immune cells interactions in the TME. Expression of specific cell markers and assessment of cell proliferation were carried out to characterize the proposed 3D co-culture model. Additionally, the study and profiling of the secretome revealed a subset of particular cancer-related inflammation proteins prompted upon 3D cultivation of tumor cells in presence of lymphocytes, pointing out an intercellular communication. Altogether, these results suggest that our 3D cell co-culture model can be a useful tool to identify and study critical factors mediating the crosstalk between tumor and immune cells in the TME. Finally, the potential of this model as a drug-screening platform has been explored using docetaxel as a model antitumoral compound.